XENOBIOTIC EFFECTS ON GENE EXPRESSION IN ENDOMETRIAL CELLS AND
GRANTLEY D. CHARLES
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
This dissertation is dedicated to the memory of my mother
VALERIE LOORJN CHARLES
I would like to thank first and foremost my mentor, Dr. Kathleen Shiverick, for
guiding me during the course of my research and for refusing to accept anything less than
my best. I would also like to thank the present and past members of the laboratory, Dr.
Phyllis Conliffe, Dr. Scott Masten, Dr. Mary Vaccarello, Dr. Liyan Zhang, Dr. Yara Smit,
Paul Saunders and most of all Theresa Medrano, for having made this not only a great
working environment, but a fun and entertaining one as well.
I would like to express my thanks to my fellow graduate students, Sam, Alan and
Srini, for their friendship and camaraderie during our study together over the years. I also
wish to thank the members of my dissertation committee. Dr. William Buhi, Dr. William
Farmerie, Dr. Jeffrey Harrison, Dr. Thomas Rowe and Dr. David Silverman, for the ready
access to their advice and support during the course of my work. I am also grateful to the
remaining faculty and staff of the Department of Pharmacology for having made my time
here both rewarding and pleasant.
I would like to acknowledge the following people for the contribution of materials
required for the completion of this work: Dr. Thomas Rowe, Dr. WiUiam Buhi, Dr.
WiUiam Greenlee, Dr. Daniel Linzer, Dr. Mary Duckworth and Dr. Michael Soares.
Especial thanks go to Dr. Maria Grant and Dr. William Farmerie not only for the material
and support provided during the course of my research, but for the personal guidance and
instruction they provided.
Finally, I would like to thank my father, sisters and my uncle and his family for
their support when the light at the end of the tunnel seemed to distant to attain.
TABLE OF CONTENTS
KEY TO ABBREVIATIONS vii
CHAPTER 1: INTRODUCTION 1
Study Objectives 1
Uterine Disease: Association with Xenobiotic Agents 2
Endometriosis: Disease Histogenesis, Etiology and Promotion 5
Mechanisms of Action of TCDD and B(a)P 7
Ah Receptor Activity 7
AhR Independent Mechanisms 8
Use of Endometrial Carcinoma Cells for the Study of the Potential Role of
Xenobiotics in Endometriosis 9
PRL-GH Related Protein Expression in Rodent Placental Tissue 10
Placental Prolactin Family of Proteins 10
Biological Activities of Placental Proteins 12
Angiogenesis, Xenobiotics and Placental-Fetal Development 14
CHAPTER 2: MATERIALS AND METHODS 16
Chemicals and Bioreagents 16
Recombinant cDNA Clones, Vectors and Plasmids 17
Antibodies and Antisera Generation 18
Cell Culture and Chemical Treatments 18
RL95-2 Endometrial Carcinoma Cells 18
Nb2 Cells 19
Chinese Hamster Ovary (CHO) Ceils 19
Retinal Endothelial Cells 19
Expression and Purification of hCAII-PLP-C Fusion Protein 20
Construction of the pET22b(+)PLP-C Bacterial Expression System.21
Expression Vector 21
Purification of Recombinant His-PLP-C 21
Amino-terminal Sequence Analysis 22
Construction of PLP-B Mammalian Expression System 22
Expression Vector 22
Transfection and Stable Mammalian Expression of PLP-B .. 23
Basal Zone Explant Conditioned Media 24
Immuno-Affinity Protein Purification 24
Endothelial Cell Migration Assay 25
In Vitro Invasion Assay 25
Nb2 Assay for Lactogenic Activity 26
EGFR Binding Assay 27
Western Immunoblot Analysis 27
General Proceure 27
EOF Receptor and CYPlAl Protein 28
PLP-B and PLP-C Protein 28
RNA Isolation & Northern Blot Analysis 28
Nuclear Run Off Assay 29
Fibrin Zymography 30
Data Analysis 31
CHAPTER 3: EFFECTS OF TCDD AND B(a)P ON CELLULAR
PROLIFERATION AND EOF RECEPTOR EXPRESSION
IN THE RL95-2 CELL LINE 32
Effects of TCDD and B(a)P on CYPlAl and CYPIBI
mRNA in RL95-2 Cells 33
Effects of TCDD on "^I-EGF Binding in RL95-2 Cells 34
Effects of TCDD and B(a)P on EGFR and CYPlAl
Protein in RL95-2 Cells 34
Effects of TCDD and B(a)P on Cellular Proliferation
in RL95-2 Cells 35
Effect of TCDD and B(a)P on Steady State c-myc
mRNA Levels 36
CHAPTER 4: EFFECTS OF TCDD AND B(a)P ON CELLULAR
INVASIVENESS AND THE EXPRESSION OF uPA AND
TIMPs IN A HUMAN ENDOMETRIAL CELL LINE 47
Evaluation of the Effect of TCDD and B(a)P on
RL95-2 Cellular Invasiveness 49
Effect of TCDD and B(a)P on uPA mRNA SteadyState
Levels and Plasminogen Activity in RL95-2 Cells ... 50
Effects of TCDD and B(a)P on TIMP mRNA Expression
in RL95-2 Cells 51
CHAPTER 5: EFFECTS OF TCDD ON IL-lp AND TNFa IN A HUMAN
ENDOMETRIAL CELL LINE 66
Effects of TCDD on IL-ip and TNF-a mRNA levels
in RL95-2 Cells 67
Effect of TCDD on the Rate of Transcription of CYPlAl,
CYPIBI, uPA, and IL-ip mRNA in RL95-2 Cells ..68
CHAPTER 6: EXPRESSION AND PURIFICATION OF RECOMBINANT
AND NATIVE PROLACTIN-LIKE PROTEINS B AND C 78
Purification of Recombinant hCAII-PLP-C 79
Recombinant Expression and Purification of
Expression of Recombinant PLP-B 82
Purification and Western Immunoblot Analysis of Native
PLP-B and PLP-C 83
CHAPTER 7: CHARACTERIZATION OF NATIVE AND RECOMBINANT
PROLACTIN-LIKE PROTEINS B AND C 100
Effects of Native PLP-B and PLP-C and Recombinant
PLP-C on Nb2 Lymphoma Cell Proliferation 101
Effects of Placental Conditioned Medium, and Native
PLP-B and PLP-C on Endothelial Cell Migration .... 101
CHAPTER 8 : CONCLUSIONS AND FUTURE DIRECTIONS Ill
LIST OF REFERENCES 117
BIOGRAPHICAL SKETCH 136
KEY TO ABBREVIATIONS
aryl hydrocarbon receptor
aryl hydrocarbon nuclear translocator
bovine brain endothelial
bovine serum albumin
cellular myc ribonucleic acid
complementary deoxyribonucleic acid
calf intestinal alkaline phosphatase
cytochrome P450 lAl
cytochrome P450 IBl
dulbecco's modified eagle's medium
dioxin responsive element/xenobiotic responsive element
epidermal growth factor
fetal bovine serum
gonadotropin releasing hormone
human carbonic anhydrase II
heat shock protein
insulin-like growth factorII/mannose-6-phosphate
messenger ribonucleic acid
protein kinase C
phenyl-methyl sulfonyl fluoride
standard error of mean
sodium dodecyl sulfate polyacrylamide gel electrophoresis
transforming growth factor
tissue inhibitor of metalloproteinase
tumor necrosis factor alpha
tissue plasminogen activator
urokinase plasminogen activator
Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
XENOBIOTIC EFFECTS ON GENE EXPRESSION IN ENDOMETRIAL CELLS AND
Grantley D. Charles
Chairman: Kathleen T. Shiverick
Major Department: Pharmacology and Therapeutics
This study evaluated, firstly, the potential role of the environmental contaminants
2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo(a)pyrene [B(a)P] in uterine disease
utilizing an endometrial adenocarcinoma cell line RL95-2. Secondly, this research study
investigated the potential lactogenic and angiogenic activity of two members of the
prolactin/growth hormone family, prolactin-like proteins B and C which are secreted by the
rat placenta during pregnancy.
TCDD and B(a)P were evaluated for their abihty to alter the expression of growth
factor and cytokine genes including epidermal growth factor (EGF) receptor, urokinase
plasminogen activator (uPA), interleukin (EL-lp) and tumor necrosis factor (TNF-a). This
study demonstrated that both TCDD and B(a)P induced the expression of CYPlAl in
RL95-2 cells, but only B(a)P significantly decreased EGF receptor expression. TCDD but
not B(a)P significantly increased the steady state level of uPA messenger ribonucleic acid
(mRNA), however neither chemical was able to significantly alter the associated fibrinolytic
activity of conditioned medium from treated RL95-2 cultures. Furthermore, TCDD
increased the mRNA expression level of TNF-a in a time-dependent and IL-1 p in a time-
and dose-dependent manner. Finally, B(a)P, but not TCDD, was able to inhibit
significantly, the overall proliferation and invasiveness of these endometrial
adenocarcinoma cultures. These results indicate that TCDD and B(a)P can alter the gene
expression of members of the growth factor/cytokine network in uterine tissue and so
potentially contribute to the promotion of uterine disease.
Native preparations of PLP-B and PLP-C purified from the conditioned medium of
placental explant cultures exhibited little lactogenic activity relative to ovine prolactin, as
evaluated by their ability to stimulate the proliferation of the rat Nb2 lymphoma cell line.
Conditioned media from gestation day 18 rat placenta significantly increased the directional
migration of human retinal endothelial cells, a measure of angiogenic activity. In contrast,
neither immunopurified native PLP-B nor PLP-C proteins showed any significant
stimulation of endothelial cell migration. Both PLP-B and PLP-C were successfully
expressed as recombinant proteins in mammalian and bacterial systems. Recombinant
PLP-C, in contrast to the native preparation, did not exhibit any measurable lactogenic
activity. In summary, the angiogenic activity exhibited by conditioned medium of rat
placental cultures is not associated with either PLP-B or PLP-C.
This research was undertaken to investigate the potential role of environmental
contaminants in uterine disease and altered placental function. The objectives of the
projects were to investigate the potential mechanisms whereby prototype xenobiotics might
(a) potentially contribute to the promotion of uterine disease in general, and endometriosis
in particular, as well as (b) alter placentaL/fetal growth and development. Firstly, recent
evidence indicates that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other
environmental pollutants such as methoxychlor (MXC) may contribute to the promotion of
uterine endometriosis, a benign proliferative disorder (Cummings and Metcalfe, 1995;
Gerhard and Runnebaum, 1992; Koninckx et al., 1994; Mayani et al., 1997; Rier et al.,
1993). It is now recognized that TCDD and other xenobiotics, which are capable of
binding to a cytosolic receptor designated the arylhydrocarbon receptor (AhR), can also act
as endocrine and growth modulators via alterations in the expression of a number of genes.
Endometriosis is an enigmatic disorder, however, with little being known regarding its
histogenesis and maintainence. Our hypothesis for this investigation is that TCDD and
benzo(a)pyrene B(a)P are able to alter the expression of uterine genes and gene products
involved in implantation and immune responsiveness which can contribute to uterine
Secondly, our laboratory has previously shown that gestational exposure of
pregnant rats to |3-napthoflavone (pNF) and 3-methylcholanthrene (3-MC), prototypical
polyaromatic hydrocarbons (PAH) and AhR ligands, resulted in intrauterine fetal growth
retardation (lUGR) and decreased placental function (Fuhrman-Lane et al., 1983; Shiverick
et al., 1984). Furthermore, PNF treatment was found to be associated with the decreased
secretion of a family of placental prolactin-like proteins (PLPs) into the conditioned media
of placental explant cultures (Shiverick et al., 1991). These rat placental prolactin-like
proteins have become of particular interest based upon the recent demonstration of the
angiogenic and angiolytic activity of two potentially homologous proteins in murine
placenta (Jackson et al., 1994). Thus, these data led us to hypothesize that pNF-mediated
alteration in the expression of rat placental PLPs could result in a decreased ability of the
placenta to develop a vascular network capable of maintaining adequate fetal growth. To
investigate this possibility, we purified native rat placental PLP-B and PLP-C as well as
expressed recombinant forms of these proteins in bacterial and mammalian systems in order
to evaluate them for potential lactogenic and angiogenic activity. The objective of this part
of my reseai-ch has been to elucidate the potential role(s) of these placental members of the
PRL-GH family during the course of pregnancy.
Uterine Disease: Association with Xenobiotic Agents
There has been increasing public and scientific concern that environmental
pollutants may be able to disrupt the normal hormonal milieu in humans and animals
leading to disease pathologies. Many naturally occurring and man-made chemicals present
in the environment possess estrogenic and antiestrogenic activity. These include plant and
fungal products, pesticides, plasticizers, and other industrial and agricultural chemicals
(Stancel et al., 1995). Reports of abnormal sexual development in reptiles (Guillette et al.,
1994) and birds (Fry, 1995) have provided evidence to support the proposal that select
environmental chemicals function as endocrine modulators. Concerns continue to grow
regarding the potential involvement of these agents in such reproductive abnormahties as
breast cancer, endometriosis, fibroids and uterine adenocarcinoma in women (Newbold,
The growth response of the uterus to steroid hormones is a highly regulated process
in reproductively cycling women. Hence this is a potential site for the manifestation of the
endocrine dismptive effects of environmental pollutants. Studies in rodents exposed to
diethylstilbestrol (DES), a potent synthetic estrogen, during the prenatal and neonatal
period showed epithelial and stromal stimulation of the uterine horns, cystic endometrial
hyperplasia, as well as a low incidence of benign (leiomyoma) and malignant
(adenocarcinoma) uterine tumors (Newbold et al., 1990; Newbold, 1995). This evidence
supported the conclusion that developmental exposure to estrogen affects the pattem of
uterine cell differentiation, resulting in later morphological and neoplastic alterations.
A number of environmental pollutants have been shown to modulate the growth and
differentiation of uterine tissue in both in vitro and in vivo models. The pesticides
dichlorodiphenyltrichloroethane (DDT) and MXC stimulated DNA synthesis in primary
cultures of uterine epithehal and stromal cells respectively (Tiemann et al., 1996). The
administration of o,p'-DDT to immature rats mimicked the effects of estrogen by increasing
uterine weight, as well as by stimulating DNA synthesis and cell division which led to
hyperplasia in the uterine luminal epithelium, stroma and myometrium (Kupfer, 1981;
Robison et al., 1985). Similarly, the polychlorinatedbiphenyl (PCB) Arochlor 1221 was
found to induce uterine growth in neonatally exposed rats (Gellert, 1978).
Some environmental agents, however, exhibit activity which opposes the effects of
estrogen. The characteristic example is TCDD which has been demonstrated to have
antiestrogenic properties. TCDD exposure has been linked with a variety of antiestrogenic
responses in the female rat uterus including inhibition of constitutive and 17j3-estradiol-
induced uterine wet weight increase, diminished nuclear and cytoplasmic estrogen and
progesterone receptor levels, as well as decreased epidermal growth factor (EOF) receptor
binding, EGF receptor and EGF receptor mRNA levels, and c-fos proto-oncogene mRNA
levels (Astroff et al., 1990, 1991; Romkes et al., 1987, Romkes and Safe, 1988). TCDD
has also been associated with a decreased age-related incidence of tumors of the uterus
(Kochiba et al., 1978). It should be noted, however, that low dose, short-term
admistration of TCDD did not appear to alter the uterotrophic response to exogenous
estrogen in ovariectomized rats (Shiverick and Muther, 1982). The PCB congener
3,3',4,4'-TeCB was reported to antagonize the uterotrophic effects of estradiol in the
immature female Sprague-Dawley rat (Jansen et al., 1993), while neonatal exposure of
female Wistar rats to B(a)P was found to significantiy reduce uterine estrogen receptor
density in adulthood (Csaba and Inczefi-Gonda, 1993).
It has been hypothesized that these environmental pollutants could potentially
contribute to an increased incidence of uterine disease in the female population based upon
their ability to modulate the growth and differentiation of uterine tissue. Evidence from
epidemiological and laboratory studies is beginning to implicate a number of these
environmental toxicants in the etiology of uterine disease pathologies. For example,
epidemiological data indicates that cigarette smoking is linked with a modest decrease in
risk for endometrial hyperplasia, while smoking women have a risk for endometrial cancer
less than half that of non-smokers, an effect more pronounced in postmenopausal
compared with premenopausal women (Baron et al., 1990; Baron, 1996). Epidemiological
data also suggest that there is an inverse association of cigarette smoking with uterine
fibroids and endometriosis (Baron, 1996; Cramer et al., 1986; Matorras et al., 1995).
There is presently insufficient data, however, to support a conclusion regarding the effects
of organochlorine compounds on endometrial cancer (Ahlborg et al., 1995).
Recent studies in rhesus monkeys and rodents indicate that TCDD and MXC may
act as promoters in the development of endometriosis (Cummings et al., 1996; Cummings
and Metcalfe, 1995; Rier et al., 1993). In rhesus monkeys, dietary intake of TCDD was
associated with an increased incidence and severity of endometriotic lesions (Rier et al.,
1993). In surgically-induced rodent models of endometriosis, both TCDD and MXC
supported the development and growth of the endometriotic implants (Cummings et al.,
1996; Cummings and Metcalfe, 1995). In addition, human epidemiological evidence is
accumulating to support an association between TCDD and the promotion of uterine disease
(Koninckx et al., 1994; Mayani et al., 1997). Women with endometriosis were also
reported to have increased concentrations of PCBs in their blood (Gerhard and
Runnebaum, 1992). Experiments in rhesus monkeys with the PCB Arochlor 1254,
however, concluded that the incidence and severity of endometriotic lesions observed in the
animals did not have any relation to the doses of PCB ingested during the study (Arnold et
al., 1996). Thus, the data are not conclusive and may reflect the different structure of
organochlorine compounds as well as their routes of metabolism.
Endometriosis: Disease Histogenesis. Etiology and Promotion
Endometriosis, the term first coined by Sampson (1921), is defined as the presence
of glands and stromal tissue, histologically similar to endometrium, outside the uterine
cavity and myometrium which is associated with vascularization and cellular proliferation.
The most common location of endometriosis is in the pelvis with greatest frequency
occurring in the ovaiy (Jenkins et al., 1986). The symptoms usually observed include the
presence of pelvic masses, pain, and infertility (Barbieri, 1992; Olive and Schwartz, 1993).
In fact, a recent study indicates that 80% of laparoscopics for infertihty result in diagnosis
of this disease (Thomas and Prenfiss, 1992), leading to 400,000 hysterectomies being
performed in the U.S.A in 1984 (Natl. Cen. Health Stat. 1986). Although the exact
incidence has been difficult to determine due to the asyptomatic nature of the disease which
requires laparoscopy or surgical visualization for a definitive diagnosis, a prevalence of
10% has been estimated in the general female population (Olive and Schwartz, 1993).
Evidence from epidemiological and cHnical studies, as well as work on animal
disease models, suggests that environmental agents as well as other factors may have a
significant role to play in the etiology and pathogenesis of the disease (Cramer et al., 1986;
Gerhard and Runnenbaum, 1992; Koninckx et al., 1994; Matorras et al., 1995; Mayani et
al., 1997; Rier et al., 1993). The main etiological factors appear to be exposure to estrogen
and the process of cychc shedding of the uterine lining. In this regard, endometriosis is
more common in women with short menstrual cycle lengths (< 27 days) and longer flow
periods (> 1 week) (Cramer et al, 1986; Matorras et al., 1995), while occurring
infrequently before puberty or after menopause. These studies further reported that
endometriosis was less common in smokers and in women with a low body mass index,
both factors which lead to lower endogenous estrogen stimulation. Recently, exposure to
environmental contaminants like TCDD and PCBs have also been associated with an
increased disease incidence in animal and human studies (Gerhard and Runnenbaum, 1992;
Mayani et al., 1997; Rier et al., 1993).
The physiological and molecular mechanisms whereby endometriotic tissue
develops and persists outside the uterine cavity are poorly understood and remain
controversial. Endometriosis has been described as the disease of theories, there being
several existing hypotheses as to its pathogenesis. Three main concepts predominate (as
reviewed in van der Linden, 1996; OUve and Schwartz, 1993). The in situ development
theory states that endometriosis develops in the location where it is found, thought to be the
consequence of metaplasia of peritoneal or ovarian tissue (Lauchlan, 1972). This theory
does not, however, explain why the disease occurs exclusively in women, in the pelvic
organs, during their reproductive years. The second concept, the induction theory, argues
that endometriosis results from the differentiation of mesenchyme induced by substances
released by degenerating endometrium (Merrill, 1966). The third theory of histogenesis is
that uterine endometrium is transplanted to ectopic locations through lymphatic and vascular
dissemination and retrograde menstruation (Sampson, 1927). The implantation theory is at
present the most widely held with the anatomical patterns of the disease being consistent
with retrograde menstruation (Jenkins et al., 1986). The implantation theory, however,
does not account for the fact that retrograde menstruation occurs in the vast majority of the
female population, whereas endometriosis develops in only a fraction of the these women.
This inconsistency has led to the postulate that an impaired immune response or response to
tissue injui-y may result in the inability to remove refluxed menstrual debris, thereby
increasing the possibility of endometriosis (Dmowski et al., 1994; Gleicher and Pratt,
Mechanisms of Action of TCDD and B("a)P
Ah Receptor Activity
The aryl hydrocarbon receptor (AhR) mediates most of the toxicological effects of
certain halogenated aromatic hydrocarbons that are widely disseminated in the environment,
including TCDD and polyaromatic hydrocarbons (PAHs) found in cigarette smoke and
industrial exhaust (Hankinson, 1994; Whitlock, 1993). The unUganded AhR is a basic-
heUx-loop-hehx protein which resides in the cytoplasm in association with the 90 kDa heat
shock protein (hsp90) and functions as a ligand-activated transcription factor (Hankinson,
1994; Whitlock et al., 1996). While TCDD and related PAHs like B(a)P are known to bind
the receptor, little is known regarding the binding of physiological ligands to the Ah
receptor. Compounds like indole-3-carbinol (IC3) and 3,3'-diindolylmethane (DIM) found
in cruciferous vegetables, however, have been shown to activate the AhR, although they
possess a much lower receptor affinity (Jellinck et al., 1993; Kleman et al., 1994). The
AhR has been implicated as the primary mediator of toxicity of the PAHs based on
evidence that the toxicity of individual congeners is correlated with affinity for the receptor,
as well as that susceptibility to a range of toxic effects segregates with the Ah allele in
highly responsive mouse strains like C57BL/6 (Okey et al., 1994).
The binding of ligands like TCDD is thought to result in allosteric changes in the
dimerization domain of the AhR which allows for release of the hsp90, followed by
translocation to the nucleus and interaction with a nuclear protein, the arylhydrocarbon
nuclear translocator (Arnt). There is some evidence which implicates protein kinase C
(PKC) and tyrosine kinase in the generation of an active AiiR/Arnt complex, yet the precise
role of these kinases remains unclear (Gradin et al., 1994; Schafer et al., 1993). The
heterodimeric AhR:Amt complex, possibly in association with other proteins/factors (Chan
et al., 1994; Dunn II et al., 1996), is able to bind to enhancer sequences termed dioxin or
xenobiotic response elements (DREs or XREs) upstream of structural genes such as
CYPIAI. Xenobiotic-induced binding of the AhR/Amt complex to enhancer chromatin
sequences is then associated with localized changes in chromatin structure manifested by
increased accessibility of the gene promoter DNA sequence to general transcription factors
such as the TATA-binding protein. The initiation complex formed at the promoter then
allows for gene transcription to be initiated (Okino and Whitlock, 1995; Whitlock et al.,
The principal route of AhR mediated toxicity is thought to occur via the ability of
the AhR/Amt complex to facihtate the increased transcription of metabolic enzymes like
CYPlAl leading to the bioactivation of pretoxicants to their reactive metabolites, which
may result in cytotoxic, carcinogenic or teratogenic effects. In addition, these regulated
enzymes may also play a role in the metabolism of endogenous compounds involved in the
control of cellular growth and differentiation (Hankinson, 1994; Okey et al., 1994). The
fact that TCDD, B(a)P and related compounds have been demonstrated to alter gene
expression in uterine tissue, coupled with the ubiquity of AhR tissue expression (Dolwick
et al., 1993), makes it feasible to investigate the potential action of these compounds in the
promotion of uterine pathologies.
AhR Independent Mechanisms
Some scientists question that AhR-mediated induction of gene transcription is the
sole route for the toxic effects of TCDD and related compounds. Recent work under cell-
free conditions has demonstrated that TCDD is able to activate protein kinases in the
absence of a nucleus (Enan and Matsumura, 1995). Induction of immediate-early response
protooncogenes like c-fos andjwnB by TCDD and B(a)P appears to be independent of AhR
and Arnt in variant hepatoma cell lines (Hoffer et al., 1996; Puga et al., 1992). This
evidence lends support to the possibility of distinct signal transduction pathways for the
mediation of the toxic effects of these compounds. Furthermore, the toxic effects of B(a)P
may be exacerbated by metabohsm in mammalian cells to reactive products that can
covalently bind to DNA, as well as through the generation of reactive oxygen species
capable of producing direct cellular damage (Leadon et al., 1988). TCDD is itself highly
resistant to metabolism and has an approximate 7-10 year half life in humans. These
differences in mechanisms of action may account for the observed differences in the
epidemiological findings of cigarette smoking compared with TCDD as relates to uterine
disease (Ahlborg et al., 1995). Our study of their comparative effects in our endometrial
culture system should further the understanding of these empirical observations.
Use of Endometrial Carcinoma Cells for the Study of the Potential Role of Xenobintics in
The question sometimes arises as to the appropriateness of utilizing in vitro cultures of
transformed cells as models for the study of disease pathologies. In this regard, a major
concern is the potential difference in responsiveness of transformed cells compared to the
original uterine tissue. The use of primary endometrial cell culture, however, presents a
number of problems, such as the cyclical hormonal variation which exists throughout the
menstiual cycle, the lack of tissue homogeneity, finite life span, interindividual variation of
the tissue, and potential loss of steroid and other receptor signalling systems (Tabibzadeh et
al., 1990; Watson et al., 1994). The use of tissue from patients with endometriosis
presents similar difficulties with patient variabihty in severity of this disease, as well as that
of the availability of a reliable supply of tissue for culture maintainence.
The endometrial adrenocarcinoma RL95-2 cell line was established by Way et al
(1983) and has been investigated with respect to the action of several growth factors
including EGF, TGFa and TGF|3 (Korc et al., 1986, 1987). Liu and Teng (1994)
demonstrated that estrogen was able to produce measurable responses in the RL95-2 cell
line. Similarly, Grenman et al (1988) demonstrated RL95-2 responsiveness to both
estrogen and progesterone. RL95-2 also exhibits significant EGF and TGFp binding
activity (Dumontet al., 1995; Korc et al., 1986; Lelle et al., 1993). Endometriotic tissue
from patients transcends the clinico-pathologic distinction between a benign disease and an
invasive neoplasm. While histologicaly benign, endometriotic tissue invades local pelvic
structures (Koninckx and Martin, 1992). Hence we considered the use of the
adenocarcinoma RL95-2 cell line to be appropriate as an in vitro model for the purposes of
the present study.
I PRL/GH Related Rrotein Expression in Rodent Placental Tissue
Placental Prolactin Family of Proteins
The rodent develops two distinct placental structures during the course of gestation.
The first to develop is the choriovitelline placenta which disappears by day 14 of gestation.
While the chorioviteUine placenta is degenerating, the chorioallantoic placenta comes into
existence being composed of three major regions, the labyrinth (60%), the basal or
junctional zone (15%), and the decidua basalis, subplacental region and metrial gland
(25%) which comprise the remaining part of the total placental structure late in gestation
(Davies and Glasser, 1968). The rodent placenta is a rich source of placental lactogens
(PLs) and several other proteins which have a structural homology to pituitary PRL rather
than to GH (Soares et al., 1991; Southard and Talamantes, 1991). It is predominantly in
the basal or junctional zone that the PRL-like proteins are expressed.
Amino acid or nucleotide sequence data is now available for fifteen of these
proteins. The seven placental lactogens include rat and mouse PL-I (Colosi et al., 1987a;
Robertson et al., 1990), rat PL-Iv (Deb et al., 1991; Robertson et al., 1991), rat PL-I
mosiac (Hirosawa et al., 1994), hamster, rat and mouse PL-II (Duckworth et al., 1986a;
Jackson et al., 1986; Southard et al., 1986). The seven prolactin-like proteins include:
mouse Proliferin (PLF) and Proliferin-Related Protein (PRP) (Linzer and Nathans, 1985;
Linzer et al, 1984), and rat prolactin-like proteins A, B, C, Cv and D (Dai et al., 1996;
Deb et al., 1991c; Duckworth et al., 1986b; Duckworth et al., 1988; Iwatsuki et al., 1996).
Rat decidual prolactin-related protein (dPRP), which shows a high degree of sequence
identity to PLP-C, has also been cloned from rat decidua (Roby et al., 1993), while a PRL-
like cDNA from the midgestation hamster placenta similar to PL-I has recently been isolated
and characterized (Barnes and Renegar, 1996). These proteins exhibit both cell and
temporal specific patterns of expression, being secreted predominantly by the
spongiotrophoblast and trophoblast giant cells of the junctional or basal zone.
Comparison of the amino acid sequences of these proteins indicates that amongst
the PL-Is there is over 70% sequence identity and sinndlar homology exists among the PL-
IIs. In contrast, the prolactin-like proteins A, B, C, Cv, D, Prohferin (PLF) and Prohferin
Related Protein (PRP) have between 12-40% sequence identity with each other and to the
PLs (Iwatsuki et al., 1996; Southard and Talamantes, 1991). The structural assignment of
these proteins to the PRL family has been based on the positioning of conserved cysteine
residues, as well as additional amino acid sequence homologies. Any similarities in
biological activities to PRL were not considered a requirement for inclusion, since in most
cases these activities were yet to be determined. Therefore, as a group they bear at
maximum a 45% sequence homology to pituitary PRL.
Prolactin is known to exhibit a broad range of distinct physiological actions
important in reproduction. These include regulation of amniotic fluid volume and ion
content, development of the mammary gland and milk protein production, along with
suppressing the immune response and uterine contractility (Handwerger et al., 1992). At
the onset of pregnancy, pituitary PRL secretion exhibits a twice daily surge which abruptly
terminates at midgestation when the chorioallantoic placenta develops and secretion remains
depressed for the rest of gestation (Smith and Neill, 1976). The presence of pituitary PRL
after day 6 of gestation is not required for the maintenance of the corpus luteum (Morishige
and Rothchild, 1974). It has also been observed that removal of the anterior pituitary in the
rat after midgestation did not interupt pregnancy (Pencharz and Long, 1931). Pituitary
PRL could not be replaced by decidual PRL in rodents, as it may be in humans, since
rodent decidua does not express this hormone (Handwerger et al., 1984). The secretion by
the placenta of high levels of placental lactogens and prolactin-related proteins during the
course of pregnancy has led to the hypothesis that they are of importance in the placental-
fetal growth axis.
Biological Activities of Placental Proteins
There has been a great deal of interest in determining whether or not these newly
discovered proteins were "PRL-like" in their ability to bind the PRL receptor, or in the
expression of similar bioactivities. Studies characterizing these rodent placental proteins
have demonstrated that some of them do in fact exhibit PRL-like activities. This has been
shown by the abihty of haPL-II, rat and mouse PL-I and PL-II and rat PL-Iv to exhibit
lactogenic activity by stimulating prolactin-like responses in rat Nb2 lymphoma cells
(Cohick et al., 1995; Colosi et al., 1987a, b; Deb et al., 1991c; Robertson et al., 1982,
1994), in mammary gland epitheUal cell differentiation (Soares et al., 1983; Southard et al.,
1986), and in the pigeon crop sac assay (Colosi et al., 1982). Most of the other prolactin-
like members of the family have all been expressed, but evidence does not show lactogenic
activity (Cohick et al., 1997; Conhffe et al., 1994; Rasmussen et al., 1996). Mouse PL-1
and PL-II have also recendy been shown to be luteotropic and to support progesterone
production in the mouse at midgestation (Galosy and Talamantes, 1995). The daily surges
of pituitary PRL have been demonstrated to be indirectly inhibited by placental lactogen
(Tomoganeet al., 1992). The hypothesis thus arose that this fanoily of secreted placental
members of the PRL-GH family could somehow, perhaps via a feedback mechanism,
replace PRL functionally during gestation.
Two members of the placental PRL-GH family, proliferin (PLF) and proliferin
related protein (PRP), have recently been shown to display angiogenic and angiolytic
activity, respectively (Jackson et al, 1994). PLF and PRP have also been shown to
compete with 16K PRL for binding to membranes of bovine brain endothelial (BBE) cells
(Clapp and Weiner, 1992). The 14K and 16K forms of PRL, enzymatically generated N-
terminal fragments of 23K pituitary PRL, are themselves potent angiolytic factors in BBE
ceUs (Clapp et al., 1993, 1994; Ferrara et al., 1991). Furthermore, PLF has been
demonstrated to bind to capillary endothelial cells in the placenta (Jackson et al., 1994), as
well as to sites in the developing embryonic vertebral and vascular structures (Jackson and
Linzer, 1997). Competition and comparative binding studies indicate that the insulin-like
growth factorIFmannose-6-phosphate receptor is involved in PLF binding (Lee and
Nathans, 1988; Volpert et al., 1996), as well as to a receptor in uterine membrane
preparations of pregnant mice (Nelson et al., 1995). The chemotaxis initiated by PLF and
mediated by the IGF-II/mannose-6-phosphate (IGF-II/M6P) receptor appears to occur
through a G protein-coupled pathway via MAPK activation. This was demonstrated by the
fact that PLF stimulated MAPK activity and endothelial cell chemotaxis, both activities
being blocked by pertussis toxin and the specific inhibitor of MAPK kinase, PD 098059
(Groskopf et al., 1997). Although PLP-A, PLP-B, PLP-C and dPRP have been expressed
I as recombinant proteins, their biological role(s) remain to be elucidated (Cohick et al.,
j 1997; Conhffe et al., 1994; Deb et al., 1993; Rasmussen et al., 1996).
Our laboratory has previously characterized the expression of a number of these
" placental prolactin-like proteins in the rat placenta (Ogilvie et al., 1990a, b). Observations
from our laboratory have associated matemal xenobiotic exposure and protein malnutrition
with the decreased secretion of placental prolactin-like proteins from basal zone explant
cultures (Conliffe et al., 1995; Shiverick et al., 1991). A further correlation was noted
with decreased placental vascularization and intrauterine growth retardation which has led
us to further investigate the potential for two of these proteins, PLP-B and PLP-C, to
exhibit lactogenic and angiogenic activity. Although they may not be the homologs of PLF
and PRP, they may function similarly, although they appear to lack the conserved amino
acid residues essential for lactogenic activity (de Vos et al., 1992; Goffin et al., 1993,
1994, 1996; Somers et al., 1994; Southard and Talamantes, 1991). Angiogenesis is an
important factor in placental development. Placental vascular growth begins early in
pregnancy and continues throughout gestation, with dramatic increases in fetal-maternal
blood flow. Similarly, placental anti-angiogenic factors likely target the maternal placental
vasculature and may function to limit vascular development and possible invasion by fetal
tissue. Therefore the potential for PLP-B and PLP-C to act as angiogenic or angiolytic
factors warrants investigation.
Angiogenesis. Xenobiotics and Placental-Fetal Development
Angiogenesis was first coined as a term to describe the formation of new blood
vessels in the placenta (Hertig, 1935). It is the biological mechanism of new capillary
formation involving the activation, migration and proliferation of endothelial cells from pre-
existing venules (Hockel et al., 1993). Defects in angiogenesis may contribute to a variety
of disorders such as endometrial hyperplasia, dysfunctional uterine bleeding,
endometriosis, pregnancy loss, pre-eclampsia and cancer (Gordon et al., 1995). The
process of angiogenesis has a critical role to play in placental/fetal establishment and
development (Reynolds et al., 1992; Welsh and Enders, 1991). Throughout gestation,
placental transport capacity keeps pace with fetal growth and uterine blood flow increases
approximately three to four-fold from mid to late gestation (Reynolds et al., 1986).
Consequently, factors which contribute to inadequate placental vascular development may
have a tremendous impact on fetal growth and development, and, ultimately, on neonatal
growth and survival. Embryonic wastage and reduced birth weights are recognized to be
major socio-economic problems associated with pregnancy (Reynolds and Redmer, 1995).
The association of environmental agents like cigarette smoke, TCDD and PCBs
with the incidence of teratogenicity, low birth weight and fetotoxicity (Couture et al., 1990;
McNulty, 1985; Sachs, 1989) may be related to their ability to interfere with the
neovascularization process during the course of gestation. Evidence to support this
hypothesis has begun to accumulate. For example, pregnant mice exposed to TCDD and
2,3,4,7, 8-pentachlorodibenzofuran (4-PeCDF) exhibit rupture of the embryo-maternal
vascular barrier and the visceral yolk sac membrane, resulting in the hemorrhaging of
embryonic blood into the maternal circulation and the uterine and amniotic cavities (Khera,
1992). Similarly, TCDD exposure in bird and fish models leads to pericardial and yolk sac
edema and hemorrhaging (Henry et al., 1997; Spitsbergen et al., 1991).
The exact mechanism underlying this pathology is not well understood, but appears
to be, at least in part, due to the ability of these agents to cause vascular derangements,
possibly via disruption of endothelial cell barrier function, or alternatively, as a result of
CYPlAl induction with resulting oxidative damage (Guiney et al., 1997; Stegeman et al.,
1995; Toborek et al., 1995). The possibility that these environmental pollutants may also
act through endocrine disruptive mechanisms is highlighted by the fact that functional
estrogen receptors are required for the augmentation of basic Fibroblast Growth Factor
(bFGF)-induced uterine angiogenesis in female mice (Johns et al., 1996). The effect
appears to be an AhR-mediated process as seen by the fact that those compounds which do
not bind the AhR, do not produce endothelial cell dysfunction (Toborek et al., 1995). In
addition, Arnt " embryos die in utero as a consequence of abnormal yolk sac angiogenesis
(Maltepe et al., 1997). Finally, CYPIA induction in endothelium in early development in a
trout model appears to correlate with mortality and the development of edemas prior to
death, a process which is consistent with involvement of the AhR (Guiney et al, 1997).
MATERIALS AND METHODS
Chemicals and Bioreagents
TCDD was obtained from Midwest Research Institute (Kansas City, MO) through
the National Cancer Institute Chemical Carcinogen Reference Repository. Benzo(a)pyrene,
aminopterin, insulin, transferrin, endothelial cell growth supplement and ampicillin were
purchased from the Sigma Chemical Co. (St. Louis, MO). Ovine prolactin was obtained
from the National Hormone and Pituitary Program (NHPP). ['^^I]-EGF and [^H]-methyl-
thymidine were purchased from Amersham Life Sciences (ArUngton Heights, IL) and [a-
^^PJdCTP and [a-'-P]UTP from ICN Biomedicals Inc. (Irvine, CA). The Prime It® II
random primer labelling kit and Nuctrap® probe purification columns were purchased from
Strategene (La loUa, CA) and ExpressHyb hybridization buffer from Clontech Laboratories
Inc. (Palo Alto, CA). The CellTiter 96^^ nonisotopic cell proliferation assay kit was
obtained from Promega (Madison, WI). Restriction and modifying enzymes were
purchased from Promega (Madison, WI), New England Biolabs (Beverly, CA) or
Gibco/BRL (Grand Island, NY). The Fisher Leukostat^M stain was from Fisher Scientific
(Lexington, MA). p-Aminomethylbenzenesulfonamide agarose resin (pAMBS) was
purchased from Sigma Chemical Co. (St. Louis, MO) and ProBond™ Resin from
Invitrogen (San Diego, CA). Cell culture media, Lipofectin® and antibiotics were from Life
Technologies (Gaithersburg, MD) and Sigma Chemical Co (St. Louis, MO), with the
exception of fetal bovine serum which was obtained from Hyclone Laboratories (Logan
UT). Plasminogen, thrombin and fibrinogen were purchased from CALBIOCHEM®
Biochemicals (La JoUa, CA). Low gelling temperature Sea Plaque Agarose^M was obtained
from FMC® BioProducts (Rockland, ME), enterokinase from Biozyme Laboratories
International Ltd. (San Diego, CA), G418 sulphate from CELLGRO (Herndon, VA), and
bovine dermal collagen and Matrigel from Collaborative Biomedical Products (Bedford,
MA). All other chemicals were reagent or molecular biology grade and were obtained from
standard commercial sources.
Recombinant cDNA Clones. Vectors and Plasmids
Plasmids containing cDNA for EOF receptor (pE7), TGF-a (phTGFl-10-3350),
c-myc (pGl-5'-c-myc), CYPlAl (phPl-450-3'), uPA (pHUK-8), TIMP-1 (pALP-181-
EPA57), TIMP-2 (pSS38), TNF-a (pAW739) and P-actin (HHCI89; 65128) were
obtained from the American Tissue Type Culture Collection (ATCC) (Rockville, MD). The
plasmids containing cDNA for CYPIBI (Sutter et al., 1994) and DL-lp (Sutter et al., 1991)
were kindly provided by Dr. William Greenlee (University of Massachusetts, Worchester,
MA). Full length cDNAs for PLP-B and PLP-C (Clone C-308) were generous gifts of Dr.
Mary Duckworth (University of Manitoba, Canada) and Dr. Michael Soares (University of
Kansas, Kansas City, KS), respectively. For Northern blot analyses, the probes used
were a 1.0 kb EcoRL fragment for CYPlAl, a 2.4 kb CM fragment for EGF receptor, a
1.6 kb Sad fragment for c-myc, a 1.5 kb Pstl fragment for uPA, a 410 bp AvaVHincU
fragment for TIMP-1, a 790 bp EcoRJ/Xbal fragment for TIMP-2, a 1.5 kb Eagl fragment
for CYPIBI, a 1.4 kb Eagl fragment for IL-lp, a 1.3 kb Hindlll fragment for TNFa and
a 1.1 kb EcoRl fragment for P-Actin. The mammalian expression vector pMXSND (Lee
and Nathans 1988) was generously provided by Dr. Daniel Linzer (Northwestern
University, Evanston, IL). The bacterial expression vector pET22b(-l-) (Novagen, San
Diego, CA) and all other non-expressing bacterial strains were provided by Dr. William
Farmerie and G. Van Heeke (University of Florida, Interdisciplinary Center for
Biotechnology Research [ICBR], Protein Expression Core, Gainesville, FL).
Oligonucleotide primers were synthesized by the ICBR DNA Synthesis Core.
Antibodies and Antisera Generation
A polyclonal sheep anti-human EGF receptor antiserum was acquired from Upstate
Biotechnology Inc. (Lake Placid, NY) and the polyclonal goat anti-rat CYPl Al antiserum
from Gentest (Woburn, MA). The horseradish peroxidase labeled goat anti-sheep IgG and
rabbit anti-goat IgG were purchased from Bio-Rad Laboratories (Hercules, CA). Rabbit
polyclonal antisera against PLP-B was previously generated in our laboratory (Ogilvie et
al., 1990a). To generate antiserum against recombinant PLP-C, a New Zealand White
rabbit was injected subcutaneously with 220 [ig of purified recombinant PLP-C in Freund's
complete adjuvant, and three booster injections of 300 |j,g hCAII-PLP-C fusion in Freund's
incomplete adjuvant were administered at 1 week intervals. Two weeks after the final
booster, the animal was bled and hCAIE cross-reactivity was absorbed out during an
overnight incubation with hCAII-pAMBS resin. The hCAH-pAMBS resin was generated
by incubating hCAII with pAMBS overnight.
Cell Cultures and Chemical Treatments
RL95-2 Endometrial Carcinoma Cells The human endometrial carcinoma cell hne RL95-2
was obtained from ATCC and maintained in DMEM:HAM'S F-12 (1:1) supplemented with
10% (w/v) FBS in a humidified atmosphere containing 5% CO? at 37°C. All media
contained penicillin and streptomycin at 100 p.g/ml. Media was changed every 2-3 days,
and all experiments initiated when cells were at approximately 50-75% confluence. Cells
were cultured in the presence or absence of chemical treatments, added in either DMSO,
ethanol or buffered aqueous solution. Stock solutions of TCDD and B(a)P were prepared
in DMSO and added to cultures with final DMSO concentrations at 0.1% (w/v).
Appropriate vehicles were added to cultures as controls.
Nb2 cells The Nb2-1 Ic subline was kindly provided by Dr. Paul Kelly (INSERM, Paris,
France). It was maintained in RPMI 1640 media supplemented with 100 U/ml penicillin,
100 |ig/ml streptomycin (GIBCOBRL, Grand Island, NY), 50 |.iM 2-mercaptoethanol,
10% (w/v) horse serum (HS) and 10% (w/v) FBS. Starvation media was composed of the
maintainence media minus FBS.
Chinese Hamster Ovarv (CUO) Cells The CHO cell line was obtained from ATCC and
routinely maintained in HAM'S F-12 supplemented with 100 U/ml penicillin, 100 f^g/ml
streptomycin and 10% (w/v) FBS. For the collection of recombinant PLP-B, the stably
transfected cells were transferred to HAM'S F-12 with 25-40 nM CdCl,, and conditioned
media aspirated every 24-48 hr and stored at -20°C.
Retinal EndotheUal Cells Human capillary endothelial cells were isolated from
collagenase-digested donor retinas using the technique described by del Vecchio and
Schaffer (1991) and modified as in Grant and Guay (1991). The purity of the primary
endothelial cell cultures was evaluated by phase contrast microcopy and immunoflourescent
labeling with acetylated low-density lipoprotein labeled with l,rdioctadycl-3,3,3',3'
tetramethyl-indocarbocyanin perchlorate (Dil-Ac-LDL, Biomedical Technologies,
Stoughton, MA). Cells from passage 5-7 were used in the migration stiidies. The primary
endothelial cultures were grown on 0.2% (w/v) gelatin coated plates and maintained in
DMEMwith 10% (w/v) FBS, 0.5 ^ig/ml insulin and tiansferrin, 100 p,g/ml streptomycin,
100 U/ml penicillin, 0.5 |ig/ml Amphotericin B and 0.15 |ig/ml endothelial cell growth
Expression and Purification of the hCAII-PLP-C Fusion Protein
Plasmid p0304/PLP-C23 was transformed into the bacterial expression host
JM109(DE3) according to the procedure of Hanahan (1993). Cultures containing
p0304/PLP-C23 were grown at 370C overnight in Luria Broth supplemented with
ampicillin (50 !ig/ml). The overnight culture was diluted 1:500 in fresh media and grown
at 370c until the optical density measured at 550 nm reached 0.6-0.8. Expression of the
fusion protein was induced by adding 0.1 mM isopropyl (3-thiogalactosidase and 12.5 [xM
ZnCl2. Following a 5 hr incubation at room temperature, cells were harvested by
centrifugation at 1500 xg for 15 min and the cell pellet was stored at -80°C.
Cells were lysed by freeze-thawing and sonication and resuspended in cold 50 mM
Tris/0.5 mM EDTA (pH 7.8) containing O.lmM PMSF and 25 |iM ZnCl2. The suspension
was incubated with DNasel for 1 hr and then centrifuged for 30 min at 15000 x g . The
supernatant was adjusted to pH 8.7 and incubated with 3 ml pAMBS agarose resin
overnight. pAMBS affinity chromatography utilizes hCAII as the purification ligand. The
resin was recovered by centrifugation and washed with 30 bed volumes of 0. 1 M Tris/0.2
M K2SO4/O.5 mM EDTA (pH 9.0) followed by the same buffer at pH 7.0 until the
absorbance at 260 and 280 nm was effectively zero. The hCAII-PLP-C fusion protein was
eluted from the resin with 0.4 M KSCN/0.1 M Tris/0.5 mM EDTA (pH 6.8), and
concentrated using centriprep-10 concentrators (Amicon). The retentate was dialysed for
3-4 days against 1000 volumes of 50 mM Tris/2 mM Ca2+ (pH 8). All purification steps
were performed at 4°C. Protein concentration of all samples was determined by the method
of Bradford (1976).
The dialysed protein was digested (lU enterokinase:3 [ig protein) at 37''C for 30-
36 hr, and the mixture was then incubated with the pAMBS agarose resin overnight. The
resin was collected by cenUifugation and the supernatant containing recombinant PLP-C
was concentrated, dialysed and stored at -20°C.
Construction of the pET22b(+)PLP-C Bacterial Expression System
Expression Vector: The expression construct was generated by modification of the
original hCAII-PLP-C fusion construct. The hCAII-PLP-C plasmid construct was double
digested with Eael/HincE to generate a 751 bp fragment containing the PLP-C cDNA
insert. The bacterial expression vector pET22b(+) was digested with Ndel/Notl. Both
fragments were gel purified on 1% (w/v) agarose and the bands isolated with the Quiaex
gel extraction kit (Quiagen, Chatsworth, CA). Insertion of the PLP-C DNA into the
pET22b(+) system with a polyhistidine linker region required that oligonucleotide adaptors !
containing the polyhistidine sequence be annealed to the 5' end of the PLP-C insert. i
Upper Primer: 46 mer 5'- TAT GGG CCA TCA TCA TCA TCA TCA TCA
TCA TCA TCA CGT GAG CGG C -3'. Lower Primer: 44 mer 5'- GCC GCT Ij
CAC GTG ATG ATG ATG ATG ATG ATG ATG ATG ATG GCC CA -3'. To faciUtate
this procedure a three way ligation reaction was set up with the 750 bp PLP-C insert,
pET22b(+) and the oligo adaptors in the presence of T4 DNA ligase overnight at 4°C. Five
|j,l of the ligation reaction was used to transform competent JM109 cells and viable
recombinant colonies were screened on LB-Amp plates for the presence of the PLP-C
insert using a Ndel digestion. A single positive clone was transformed into the bacterial
expression host BL21(DE3).
Purification of Recombinant His-PLP-C: The recombinant bacterial expression system
was cultured as previously described for the hCAII-PLP-C fusion protein and stored at
-20°C until ready for His-PLP-C purification. The bacterial pellets were lysed in 20 mM
Tris, 5 mM Imidazole, 0.5 M NaCl (pH 7.9) containing 0.1 mM PMSF. The suspension
was incubated with DNasel for 1 hr, and then centrifuged for 30 min at 15000 x g.
Recombinant His-PLP-C was isolated from the supernatant via the ability of the Histidine
tag sequence on the protein to bind to immobilized divalent nickel using the His • Bind®
metal chelation resin (Novagen, San Diego, CA). The supernatant was passed over the
nickel column, followed by 30 volumes of wash buffer (60 mM Imidazole, 0.5 mM NaCl,
20 mM Tris pH 7.9). The bound protein was eluted in elution buffer (0.75 M Imidazole,
0.5 M NaCl, 20 mM Tris pH 7.9) and collected in 1ml fractions which were analysed on a
UVA^is spectrophtometer at 280 nm for the presence of protein. Protein fractions were
dialysed into 50 mM Tris, 5 mM Ca^^ (pH 7.9). The dialysed protein was incubated with
enterokinase at 37°C overnight, and centrifuged at 1500 x g for 10 min to remove any
precipitate. The supernatant containing cleaved recombinant PLP-C was then stored at
Amino-terminal Sequence Analysis
N-terminal amino acid sequence was determined by Edman degradation using a gas
phase protein sequencer at the University of Florida Interdisciplinary Center for
Biotechnology Research (ICBR) Protein Chemistry Core.
Construction of the PLP-B Mammalian Expression System
Expression Vector : The pGEM3-PLP-B clone (Duckworth et al., 1988) was doubly
digested with EcoRi/HincIL in order to release the 700 bp cDNA insert containing the
complete coding sequence for PLP-B. The digest was run on 1% (w/v) agarose gel and the
700 bp band excised from the gel with a scalpel and purified using the Quiaex gel
purification kit (Qiagen, Chatsworth, CA). pMXSND (Lee and Nathans, 1988) was
digested with Xhol and gel purified. Both cDNA framents were blunt ended by incubation
with 2 mM dNTP and 5 U of Klenow at 25°C for 20 min, then the enzyme was heat
inactivated by incubation at 75°C for 15 min. pMXSND was further dephosphorylated by
incubation with 40 U of Calf Intestinal Alkaline Phosphatase (CIAP) for 90 min at 37°C to
inhibit self ligation. Ligation between PLP-B and dephosphorylated pMXSND was set up
in a 20 |j.l volume with 4 U of T4 DNA Ligase at 4°C overnight. Ten |il of the ligation
mixture was used to transform competent JM109 cells. Viable colonies were used to
generate minicultures in Luria Broth (LB) and plasmid DNA isolated with the Wizard
Miniprep Kit (Promega, Madison, WI). Diagnostic digests were performed by digestion
with BamHI to ascertain the presence and correct orientation of the PLP-B insert. Clone 1 1
possessing the insert in the correct orientation was transformed into competent JM109 cells
and a DNA maxiprep performed using a Qiagen Maxiprep kit (Qiagen, Chatsworth, CA) to
produce transfection grade plasmid DNA.
Transfection and Stable Mammalian Expression of PLP-B: The pMXSND-PLP-B
construct was transfected into approximately 50-75% confluent CHOKl cells. Transfection
grade plasmid DNA was diluted to 1 mg/ml in ddHjO. Five ml of Lipofectin® was added
to 100 111 of OptiMEM for 45 min. pMXSND-PLP-B 1 \ig and 2 ^g were added to 100 |il
of OptiMEM and then mixed with the Lipofectin®. The mixture was allowed to sit for 15
min prior to addition of 1.8 ml of HAM'S F-12. The wells of the plates were washed with
serum-free, antibiotic-free media followed by the addition of the Lipofectin-DNA media.
I Plates were incubated at 37°C and 5% CO,. After 24 hr the media was aspirated and 4 ml
of HAM'S F-12, 10% (w/v) FBS added. Cells in each well were trypsinized 48 hr later,
diluted 1:100 into G418 selection media (HAM'S F-12, 10% (w/v) FBS supplemented
with 800 [ig/xnl G418) and plated in 10 cm dishes. Colonies were picked using a sterile
loop and placed in individual wells of a 96 well plate in selection media. After a week,
seven wells were trypsinized and passaged into each of two 10 cm culture plates in
selection media. After allowing the cells to attach for 2 days, the cells were fed with
methotrexate selection media, (10 |i.M methotrexate, 10% (w/v) dialysed FBS, 500 M-g/ml
G418, HAM'S F-12). Cultures were grown to 50-75% confluency and one of each of the
two plates treated with 50 nM CdClj for 24 hr prior to RNA isolation, to confirm the
presence of the message for PLP-B by Northern blot analysis using a ^-P-labelled PLP-B
cDNA probe. Further selection was performed using methotrexate at concentrations up to
180 |iM. Cells were washed in Hank's buffer and incubated in seaim-free HAM'S F-12
with 25 nM CdCl2 and conditioned media collected at 24-48 hr intervals and stored at -
Basal Zone Explant Conditioned Media
Timed pregnant Sprague Dawley rats were obtained from Holtzman (Madison,
WI). Animals were housed in a temperature controlled room with 12 hr light/dark cycles
and given food and water ad libitum. Animals were sacrificed on day 16-19 of gestation
under sodium pentobarbital anesthesia. The uteri were removed, and placental basal
(junctional) zones were isolated using forceps and dissecting scissors. Basal zone tissue
was minced and incubated under sterile conditions in modified Eagle's medium (175 mg
tissue/5 ml medium) for 24 hr at 37°C under 47.5% 02:2.5% CO2:50% N2. The culUire
medium was supplemented with 3 mg/ml glucose, 100 U/ml penicillin, 100 |ig/ml
streptomycin, 0.25 iig/ml amphotericin B, 0.2 U/ml insulin, 1% v/v non-essential amino
acids, 1% v/v MEM vitamins, and 1.5 lag/ml methionine (1/10 normal concentration).
After incubation the conditioned medium was centrifuged at 1000 x ^ for 10 min to remove
debris and stored at -20°C.
Immuno- Affinity Protein Purification:
The immunoaffinity columns for PLP-B and PLP-C were generated by linking
generated polyclonal antipeptide antisera (Ogilvie et al., 1990b) to an activated agarose
support matrix Affi-GeF'^ (Bio-Rad, Hercules, CA) according to the manufacturer's
instruction. For purification of native PLP-B and PLP-C, as well as recombinant PLP-B,
conditioned media at neutral pH was passed over the column and the column washed with
30 bed volumes of buffer (0.5 M NaCl, 10 mM phosphate, pH 7.0); bound protein was
eluted in 0.2 M Glycine, pH 2.8 and neutralized with K2HPO4, pH 9.2. Neutralized native
protein was concentrated using Amicon centriprep concentrators and stored at -20°C until
required. Protein concentrations were estimated by comparison with Coomassie stained
BSA standards on SDS-PAGE minigels.
Endothelial Cell Migration Assay
Endothelial cell migration assays were performed essentially as in Grant et al (1987)
using modified Boy den Chambers (Neuro Probe Inc., Cabin John, MD). Human retinal
endothelial cells were trypsinized from a T75 flask, and pelleted by centrifugation at 500 x
g for 5 min at room temperature. The cells were resuspended in Hank's buffer and washed
twice. Cells numbers were evaluated on a hemocytometer after staining with 0.25%
methylene blue solution. Approximately 7000 cells in 27 |il of serum-free growth media
were aUquoted into the lower wells of the migration chamber. The wells were overlaid
with a porous bovine dermal collagen coated membrane (5 iiim diameter pore size) and the
gasket seal and upper chamber attached. The apparatus was inverted at 37°C and 5% CO2
for 90 min to allow for cellular attachment, after which test protein solutions in serum-free
media were added in a total of 50 p,l/well in sextuplicate. 10% FES and serum-free media
were used as positive and negative controls, respectively. The apparatus was incubated
overnight at 3TC/5% CO2 to facilitate migration of attached cells. The apparatus was
disassembled, backside (cellular attachment side) scraped, and the membrane stained in
Leukostat''^ stain. The membraue was then mounted on a glass slide for evaluation by
counting the numbers of migrated cells/well under a light microscope. For migration
assays, cells were evaluated in quadruplicate wells for each treatment regimen.
In Vitro Invasion Assay
The Matrigel invasion assay was performed using the modified Boy den Chamber
apparatus as in the endothelial cell migration assay (Grant et al, 1987). RL95-2 cultures
were incubated with B(a)P and TCDD for 48 hr. Cells were trypsinized and pelleted by
centrifugation at 500 x g for 5 min. The cells were resuspended in Hank's buffer and
washed twice. Cells numbers were determined using a hemocytometer. Approximately
7000 cells in 27 |i.l of complete media in the presence of the respective chemicals were
aliquoted into the lower wells of the Boy den chamber. The wells were overlaid with a
porous Matrigel-coated polyvinyl-pyrrolidone-free polycarbonate membrane (8 micron
diameter pore size) and the gasket seal and upper chamber attached. The apparatus was
inverted at 37°C/5% CO2 for 90 min to allow for cellular attachment, after which time 50 |i,l
of complete media was added to the upper wells. The apparatus was incubated for 36 hr at
37°C/5% CO2, at which time the apparatus was disassembled, the backside of the
membrane (cellular attachment side) scraped, and the membrane stained in Leukostat^i^
stain. The membrane was then mounted on a glass slide for evaluation under a light
microscope by counting the number of cells per well which had invaded the membrane.
For these assays, cells were counted in quadruplicate wells for each treatment regimen.
Nb2 Assay for Lactogenic Activity
The Nb2 proliferation assay was performed essentially as described by Gout et al
(1980). The lactogenic response was evaluated using [^H]-thymidine incorporation and
MTT dye conversion as indicators of the growth response. Log phase Nb2 cells in
suspension were collected by centrifugation, washed, and resuspended in starvation media
overnight. Cells were again collected by centrifugation and 4 x lO'* cells in 100 |il were
added to the triplicate wells on a 96 well plate. Ovine PRL (NIDDK) in concentrations
between 0.1-100 ng/ml was added in triplicate wells in 50 |il aliquots as the standard
stimulatory response, while native and recombinant PLP-B and PLP-C were added in the
same volume. The cultures were harvested onto microfiber filter paper after a 4 hr [^H]-
thymidine pulse using a Brandel cell harvester at 48 hr (Gaithesburg, MD), and [^H]-
thymidine uptake was measured using a Beckman LS 7000 liquid scintillation counter.
Alternatively, Nb2 cells and the formazan product were solubilized at 48 hr after a 4 hr
exposure to MTT, and the absorbance measured at 595 nm on an Elisa plate reader (Adler
et al., 1994).
EGFR Binding Assay
RL95-2 cells were grown for 24-96 hr in monolayer culture in the presence of 0-50
nM TCDD in serum-free medium or in the presence of 2% (w/v) or 10% (w/v) FBS
supplemented DMEM/HAM'S F-12. Cells were washed three (3) times with cold PBS
(pH7.4), scraped from plates with a rubber policeman and pelleted by centrifagation. Fifty
|j.g of protein (whole cells) was incubated in serum-free DMEM-HAM'S F-12 containing
0.1% (w/v) BSA, with 400 pg ["^I]-EGF (8 x 10^ cpm) in the presence or absence of 100
ng unlabelled EGF for 16 hr at 4°C. Incubations were stopped and unbound radioactivity
removed by washing cells twice in cold PBS. Total bound cpm was measured using a
gamma counter. Specific binding was expressed as the difference between radioactivity
bound in the absence (total) and presence (non-specific binding) of excess unlabelled EGF.
Western Immunoblot Analysis
General Procedure: Cells were rinsed two to three times, collected with a cell scraper,
and lysed in 0.5-1 ml PBS using three freeze-thaw cycles. The membrane fraction was
obtained after centrifugation at 10000 x g for 15 min at 4°C and then resuspended in
ddHjO. Alternatively, cells were scraped into lysis buffer containing 10 mM Tris, 0.14 M
NaCl, 0.5% (w/v) Na-deoxycholate, 1% (v/v) Triton-X 100, 1 mM phenyl-methyl
sulfonyl flouride (PMSF), and 1 }xg/ml each of leupeptin and aprotinin. Cells were then
incubated with the lysis buffer for 1 hr at 4°C on an orbital shaker and the supematant was
recovered after centrifugation at 10000 x g for 15 min at 4°C. Samples of membrane or
total cell lysate protein (50-100 |ig) were then separated by 7.5% or 10% SDS-PA gels and
transferred electrophoretically to nitrocellulose filters using 25 mM Tris, 192 mM glycine
buffer at pH 8.2, with 20% (v/v) methanol according to the method of Towbin et al (1979).
EGF Receptor and CYPlAl Protein: For EGF receptor and CYPlAl protein analysis,
immunostaining was performed as previously described in Wang et al (1987). The
nitrocellulose membranes were washed in 20 mM Tris containing 0.1% (w/v) Tween 20
and 0.9% (w/v) NaCl, pH 7.5 (TTBS) for 15 min, and then 3% (w/v) gelatin for 30 min.
The membrane was then sequentially incubated with polyclonal anti-human EGF receptor
antiserum diluted to 1 [ig/25ml in TTBS without Tween 20 or preimmune sheep serum for
2 hr, followed by horseradish peroxidase labeled goat anti-sheep IgG for 60 min.
Alternatively, for CYPlAl the membrane was incubated with polyclonal goat anti-rat
CYPlAl (1:1000 dilution) or preimmune goat serum for 2 hr, followed by horseradish
peroxidase conjugated anti-goat IgG for 1 hr. Bands were visualized by incubation with 3-
amino-9-ethylcarbazole in the presence of 0.015% (v/v) hydrogen peroxide.
Lnmunoreactive bands were quantitated by scanning the nitrocellulose filters on a Microtek
ScanMaker II scanner and NIH image software.
PLP-B and PLP-C Protein: The blotted nitrocellulose membranes were blocked in 3%
gelatin for 30 min. The membrane was then sequentially incubated with either polyclonal
rabbit anti-rat PLP-B or PLP-C antiserum diluted 1:1000 in TBS or preimmune rabbit
serum for 2 hr. This was followed by incubation with horseradish peroxidase labeled goat
anti-rabbit IgG for 60 min. Immunoreactive bands were visualized by incubation with 3-
amino-9-ethylcarbazole in the presence of 0.015% hydrogen peroxide. Immunoreactive
bands were quantitated as described above.
RNA Isolation & Northern Blot Analysis
Total cellular RISfA was isolated from TCDD-treated cell cultures by acid guanidium
thiocyanate phenol-chloroform extraction according to Xie and Rothblum (1991). For
Northern blotting, 40 p.g of total cellular was denatured, fractionated on a 1.5% (w/v)
agarose formaldehyde gel, and transferred to nylon membranes. cDNA probes were
labelled with [a-^'P]-dCTP using a random primer labelling kit. Prehybridization was
carried out in commercially available ExpressHyb buffer at 68°C for 1 hr, and hybridization
in the same buffer for 2-4 hr after addition of the ''^P-labelled probe. The membranes were
washed three times in 2 X SSC, 0.1% (w/v) SDS at room temperature for 10 min, then
twice in 0.1 X SSC, 0.1% (w/v) SDS at 60-65°C for 30 min. Transcripts were then
visualized by autoradiography. Blots were later stripped and rehybridized with ^^P-labelled
cDNA for p-actin as a loading and transfer control in order to facilitate quantitation by
Nuclear Run Off Assay
Nuclei were prepared by a detergent lysis protocol (Ausubel et al., 1987). Actively
proliferating cultures of RL95-2 cells were exposed to 10 nM TCDD or 0.1% (v/v) DMSO
for 40 hr, after which the cells were washed twice with cold PBS, scraped from the plates
with a rubber policeman and pelleted by centrifugation at 500 x ^ for 5 min. The cell pellet
was resuspended by vortexing in 4 ml of NP-40 lysis buffer [10 mM Tris, 10 mM NaCl, 3
mM MgCh, 0.75% (v/v) NP-40 pH 7.4], placed on ice for 5 min, after which nuclei were
collected following a second round of centrifugation at 500 x g. This process was repeated
and nuclei resuspended in 500 \il of glycerol storage buffer [50 mM Tris, 5 mM MgClj,
0.1 mM EDTA, 40% (v/v) glycerol, pH 8.3] per 10^ nuclei. Nuclei were stored at -80°C
in 200 p-l aliquots until ready for use.
Nuclear runoff assays were performed essentially according to Srivastava et al
(1993) and Merscher et al (1993) with minor changes. Frozen nuclei (= 4 x 10^) per
reaction were thawed and mixed with an equal volume of 2 X reaction buffer (10 mM Tris,
5 mM MgCl2, 0.3 M KCl, 5 mM DTT, 0.2 mM EDTA, 4 mM ATP, GTP, CTP) and 75
fiCi [a-^^P]UTP. The reaction was allowed to proceed in a room temperature water bath
for 30 min at which time 50 U DNasel was added and the reaction allowed to continue for
5 min. After labelling, the nuclear suspension was mixed with 1.2 ml of guanidium
isothiocyanate reagent (Xie and Rothblum, 1991), 130 i^l chloroform- isoamylalcohol
(24:1), and vortexed. The reaction tube was left on ice for 10 min and the aqueous layer
collected after centrifugation at 10000 x g for 10 min and incubated with an equal volume
of isopropanol at -80°C overnight. The following day the RNA pellet was collected after
centriftagation at 10000 x g for 10 min and the pellet washed once in 70% (v/v) ethanol.
The pellet was air dried and dissolved in 0.5% SDS and 1 jil used to determine activity
using a scintillation counter. Equal amounts of cpms («10^) from DMSO control and
TCDD experiments were added to 2 ml of ExpressHyb buffer and hybridized with
j appropriate cDNAs on nylon membranes at 65°C for 48 hr. Membranes were then washed
in 2 X SSC at room temperature for 30 min, then by 0. 1 X SSC, 0. 1 % (w/v) SDS at 62''C
for 1 hr. Dot blots were visuahzed by autoradiography following 2-5 day exposure at -
80°C. X-ray films were scanned using a Microtek ScanMaker 11 scanner and spot
intensities quantitated by densitometry with each message standardized against p-actin as
Fibrin zymography was performed according to the method of Granalli-Pipemo and
Reich (1978) with modifications by Cheng et al (1991) and Nakamura et al (1995).
Briefly, RL95-2 cells were seeded into 10 cm plates at 5 x 10^ cells per plate and allowed to
grow for three days. Cultures were exposed to TCDD and B(a)P for 36 hi" and the cultures
washed in Hank's buffer before the addition of equal volumes of serum-free media to each
plate. Conditioned media was collected from control and treated cultures after 12 or 24 hr.
Thirty |i,l of CM was mixed with SDS sample buffer and run under non-reducing
conditions on a 10% SDS-PA gel. Gels were then inmiersed in 50 mM Tris, 5 mM CaClj,
2.5% (v/v) Triton X-100 three times for 15 min to remove the SDS, and then twice more
(50 mM Tris, 5 mM CaCl,, pH 8.1) for 15 min to remove the Triton X-100. To detect the
plasminogen activator activity, the poly acryl amide gel was overlaid onto a 1% (w/v) low
temperature gelling agarose gel containing 75 ill of Img/ml of plasminogen, 30 |li1 of 1
U/ml thrombin, and 10 ml of 5 mg/ml fibrinogen, in phosphate buffered saline (PBS). The
gels were incubated overnight in a humidified atmosphere at room temperature.
Plasminogen activator activity was detected as a lysed zone on the agarose gel after staining
with Coomassie brilliant blue or amido black. Bands were scanned and fibrinolytic
activities assessed by comparison of the areas of the lytic zones.
All experiments were performed at least three times at concentrations and time
points indicated unless otherwise stated. For analysis of scanned images, control lanes
were standardized to 100% and all treatments assessed relative to controls for each indiviual
experiment. For Northern blots loading was standardized to p-actin. One factor ANOVA
analysis was performed to assess the dose-response effects with significance being
determined at the p < 0.05 level. Numerical data averaged over several experiments was
represented as Mean ± Standard Error of the Mean (SEM) and the Student's t-test used to
analyse the data. Statistical analyses were performed using Microsoft Excel software
EFFECTS OF TCDD AND B(a)P ON CELLULAR PROLIFERATION AND EOF
RECEPTOR EXPRESSION IN THE RL95-2 CELL LINE
Endometriosis is the ectopic growth of endometrial tissue outside of the uterus and
has been described as a disorder which has characteristics of both a benign disease and an
invasive neoplasm (Arnold et al., 1996). It is first and foremost a proliferative disorder in
which endometriotic tissue is mislocated to and invades extrauterine sites. In view of
recent evidence implicating environmental agents hke TCDD and cigarette smoking in the
etiology of endometriosis (Cummings et al., 1996; Mattorras et al., 1995; Mayani et al.,
1997; Rier et al., 1993), our laboratory undertook an investigation as to whether or not
these environmental agents are able to directly alter the cellular proliferation of endometrial
Human uterine endometrium proliferates in response to estrogen during the course
of the female menstrual cycle. The growth and maintenance of endometriotic tissue is
known to be dependent upon estrogen as evidenced by its extreme rarity in premenarchal
giris and the association with exogenous estrogen administration in postmenopausal
women (Goodman et al., 1989). Recent evidence suggests that the action of estrogen on
the endometrium is mediated by EOF, with EOF being able to mimic the stimulatory effects
of estrogen on DNA synthesis and lactoferrin gene expression in ovariectomized mice
(Nelson et al., 1991). Estrogen also increases EOF binding and mRNA levels for the EOF
receptor in uterine tissue (Lingham et al., 1988; Mukku and Stancel, 1985).
TCDD and B(a)P have been shown to generally downregulate EGF receptor
expression in different tissues and cell lines (Astroff et al., 1990; Guy da et al., 1990;
Hudson et al., 1986; Sewall et al., 1993; Zhang et al., 1995), although upregulation of
EGF expression by TCDD has been demonstrated developmentally (Abbott and Birnbaum,
1990; Abbott et al., 1992). EGF receptor expression has been demonstrated in human
endometriotic tissue (Huang and Yeh, 1994; Prentice et al., 1992), as well as in
endometriotic lesions from surgically-induced animal models (Simms et al., 1991; Zhang et
al., 1993a). Therefore, EGF receptor expression may have a role to play in the
pathogenesis of endometriosis.
Our study evaluated the potential changes in EGF binding and EGF receptor protein
expression in endometrial cells subsequent to TCDD and B(a)P exposure. The goal of the
study was to ascertain whether or not a potential correlation could be made between EGF
receptor expression and cellular proliferation, as well as with the induction of the metabolic
enzymes CYPlAl which is classically induced by TCDD and B(a)P exposure (Sutter et al.,
1994; Whitlock, 1989).
Effects of TCDD and BfalP on CYPlAl and CYPIBI mRNA in RL95-2 Cells
We initially examined the ability of TCDD and B(a)P to induce the expression of
mRNA for CYPlAl, a classic functional biomarker of exposure to AhR agonists
(Whitlock, 1989). Northern blot analysis of 0.1% (v/v) DMSO (control) treated RL95-2
cells indicated that the 3.0 kb mRNA transcript for CYPlAl was virtually undetectable.
However, 48 hr exposure to TCDD at a concentration of 0.1 nM TCDD was able to
significantly induce CYPlAl mRNA expression with induction being maximal at the 1 nM
TCDD level (Figure 3-lA). In data not shown, induction of CYPlAl mRNA by 10 nM
TCDD was observed as early as 6 hr after exposure. Similarly, 10 [xM B(a)P exposure for
12 hr resulted in significant CYPl Al mRNA induction, with levels being maximal by 24 hi"
and maintained for the 48 hr assay period (Figure 3- IB). The expression of CYPIBI
mRNA, a second drug metabolizing enzyme recently shown to be induced by TCDD
(Sutter et al., 1994), was further analyzed by Northern blot analysis. As shown in Figure
3-lA, RL95-2 cultures express a constituitively low level of the 5.1 kb CYPIBI mRNA
transcript which is readily induced by TCDD concentrations as low as 0. 1 nM. Hence the
RL95-2 cell line appears to possess a functional AhR signal transduction system as
described in Chapter one.
Effects of TCDD on '"l-EGF Binding in RL95-2 Cells
The ability of TCDD to alter the total specific binding of '"^I-EGF to intact RL95-2
cultures was evaluated. Cultures treated with 1-100 nM TCDD for up to 96 hr did not
exhibit any significant change in the total binding of '^"^I-EGF as compared to 0.1% (v/v)
DMSO-treated controls (Figure 3-2). Total specific '^-^I-EGF binding was in the range of
10-12% as has been previously demonstrated for this cell line (Korc et al., 1986). In this
regard, it is pertinent to note that these experiments were performed at 4°C so as to
minimize internalization of the transmembrane EGF receptor.
Effects of TCDD and B(a)P on EGFR and CYPlAl Protein in RL95-2 Cells
Exposure of RL95-2 cultures to 1 nM and 10 nM TCDD showed a marked
induction of the 55 kDa CYPlAl protein band as determined by Western immunoblot
analysis (Figure 3-3A). In data not shown, this induction of immunoreactive protein was
shown to be concentration-dependent being maximal at 1 nM TCDD. Similarly, exposure
to both 1 |iM and 20 )jM B(a)P also resulted in a dose-dependent induction of CYPlAl
immunoreactive protein, although the level of induction was not as high as that exhibited by
TCDD treatment (Figure 3 -3 A). The induction of immunoreactive protein correlated with
the observed induction of mRNA transcripts for CYPlAl after exposure of RL95-2
cultures to both agents (Figure 3-1 A & B). The levels of the 170 kDa immunoreactive
EGF receptor membrane protein band were unchanged after TCDD exposure at
concentrations of 1 nM and 10 nM relative to 0.1% (v/v) DMSO controls (Figure 3-3A &
B). In data not shown, no effect of TCDD was noted for exposures up to 96 hr. By
contrast, 20 |i.M B(a)P almost totally eliminated the expression of EGFR immunoreactive
membrane protein after a 48 hr exposure period (Figure 3-3A & B). This decrease in
EGFR immunoreactive protein levels is an effect associated with B(a)P which has been
previously observed in placental choriocarcinoma cells (Zhang et al., 1995). Thus the
decrease in EGFR immunoreactive protein with B(a)P exposure was correlated with a
corresponding induction in CYPl Al protein in this cell line. In contrast, TCDD treatment
exhibited no obsei-vable decreases in EGFR protein levels while significantly greater
CYPlAl induction was observed.
Effects of TCDD and BfalP on Cellular Prohferation in RL95-2 Cells
The effects of TCDD and B(a)P on RL95-2 cell proliferation were evaluated by the
direct counting of viable cells. Under serum-free conditions, 10 |jM B(a)P significantly
decreased the rate og growth of RL95-2 cells by 48 hr after exposure (Figure 3-4).
Similarly, for cultures growing in medium containing 10% (v/v) FBS, the number of cells
in 10 |iM B(a)P-treated cultures was lower than those of 0.1% (v/v) DMSO controls by as
early as 24 hr (Figure 3-5). Furthermore, under serum-free conditions the number of
viable cells in B(a)P treated cultures appeared to plateau after 48 hr with no further increase
in cell numbers being observed for the remaining 24 hr of the assay. In contrast, 10 nM
TCDD did not result in any significant change in cell numbers compared to control cultures
for the duration of the 72 hr assay period. Thus 10 [iM B(a)P, but not 10 nM TCDD,
adversely affected cellular proliferation of RL95-2 cells under both serum-free and
complete media conditions.
Effect of TCDD and B(a)P on Steady State r-m.vc mRNA T^evels
Northern blot analysis was next used to determine whether TCDD and B(a)P
exposure of RL95-2 cultures could lead to alterations in the expression of the steady state
levels of c^myc mRNA, a proto-oncogene associated with cellular proliferation (Vasti-ik et
al., 1994). Data in Figure 3-6 show the presence of a strong constitutive level of
expression for the 2.7 kb mRNA transcript in control cells. Expression was not
significantly altered by up to 48 hr exposure to either 10 nM TCDD or 10 ^M B(a)P
(Figure 3-6A & B). Hence the decreased abihty of RL95-2 cells to proliferate in the
presence of B(a)P exposure is not correlated with a decreased level of c-myc mRNA
We chose to investigate the potential alterations in EOF receptor expression and
cellular proliferation after treatment with TCDD and B(a)P in order to gain a better
understanding of the effect these environmental agents might have on uterine growth and
uterine disease pathologies. Endometriosis is an estrogen-dependent disease (Barbieri,
1990) being rare before puberty or after menopause. Recent data indicate that estrogen
actions on the uterus may be mediated through growth factors like EOF as well as increases
in EOF receptor expression (Lingham et al., 1988; McBean et al., 1997; Nelson et al.,
1991). EGF has also been shown to be mitogenic in human endometriotic stromal tissue
(Mellor and Thomas, 1994), while experiments with rat endometriosis models suggest that
rat endometrial implants produce EGF and contain receptors for EGF (Simms et al., 1991).
Furthermore, Danazol or gonadotropin releasing hormone analogues (GnRHa), chnical
therapies for relieving the symptoms of endometriosis, significantly decrease the levels of
immunohistochemical staining for the EGF receptor (Melega et al., 1991).
The RL95-2 cell line was found to express high levels of the 170 kDa
immunoreactive EGF receptor protein in agreement with previous reports (Korc et al.,
1986, 1987; Lelle et al, 1993). In data not shown, these cells also exhibited the presence
of the 5.6 kb mRNA transcript for the EGF receptor as has been demonstrated in other
systems (Lin et al., 1991; Zhang et al., 1995). The specific binding of '"^I-EGF was not
altered by exposure to concentrations of TCDD up to 100 nM for the duration of the 96 hr
assay period compared to vehicle treated controls. This result was supported by Western
immunoblot analysis for EGF receptor, while the levels of the 5.6 kb mRNA transcript for
EGF receptor were also not significantly altered by TCDD treatment (data not shown). In
contrast to TCDD, exposure of RL95-2 cultures to B(a)P resulted in a concentration-related
decrease in the levels of the 170 kDa immunoreactive EGF receptor protein as determined
by Western analysis.
Tissue specific effects of TCDD and B(a)P on EGF receptor expression and
j binding has been a well characterized toxicological observation (Pohjanvirta and Tuomisto,
I 1994). TCDD has been demonstrated to decrease the binding capacity of EGF receptors in
the liver (Sewall et al, 1995), uterine tissue (Astroff et al., 1990) and in hepatoma cells
and keratinocytes (Hudson et al., 1985, 1986; Karenlampi et al., 1983) and, in some
.'ij cases, without a consequent decrease in steady state EGF receptor mRNA levels (Lin et al.,
1991). It should be noted that TCDD has been found to increase EGF receptor expression
during early development in the embryonic mouse palate and ureteral epithelium (Abbott
and Birnbaum, 1990; Abbott et al., 1992). In contrast, B(a)P has exhibited a similar effect
j in downregulating EGF receptor protein levels in human keratinocyte and placental cell
lines (Guyda et al., 1990; Hudson et al., 1985; Zhang et al., 1995).
The mechanism by which these compounds are able to alter EGF receptor binding
and expression are not well understood. Evidence that the effects of TCDD on the hepatic
EGF receptor may be mediated via the Ah locus was found in congenic strains of mice
differing at this locus (Lin et al, 1991). In this regard, prolonged exposure in rats
demonstrated that the EDjq for the decrease in receptor capacity was close to the EDjq for
CYPlAl induction, which is known to be regulated transcriptionally by TCDD (Sewall et
al., 1993). Furthermore, the inhibition of EGF-specific binding has been shown to be
stereospecific in that a TCDD analogue unable to bind to the Ah receptor was not able to
decrease EGF binding (Hudson et al., 1985). The results of our study indicate, however,
that both TCDD and B(a)P were able to induce the expression of both CYPlAl mRNA and
immunoreactive protein levels in the RL95-2 ceU line, while only B(a)P was able to
selectively decrease EGF receptor expression.
Karenlampi et al (1983) have proposed that electrophilic metabolites of polycyclic
aromatic hydrocarbons may be responsible for the inhibition of EGF binding observed in
mouse hepatoma cells. In this regard, TCDD is not readily metabolized and has a
significant 7-10 year half-life in humans, while B(a)P is readily metabolized to a series of
reactive species including epoxides, phenols and quinones (Gelbion, 1980). Thus the
observed down-modulation of EGF receptor protein by B(a)P may be a direct effect of
reactive metabolites which would not be observed with TCDD due to its resistance to
metabolism. Altematively, EGF receptor down-modulation can be effected by changes in
cytokines such as IL-1 and TNF (Bird and Saklatvala, 1990). As will be seen in Chapter
Five, however, TCDD is able to induce the expression of these cytokines in RL95-2 cells
without the observed decrease in EGF receptor protein.
Uterine endometrium is highly responsive to estrogen action. Evidence that the in
vivo administration of estrogen was able to stimulate c-myc expression at the transcriptional
level in rat uterus has led to the suggestion that c-myc expression is related to estrogen-
induced uterine cell proliferation (Murphy et al., 1987). Similarly, exposure of RL95-2
cell cultures to estrogen has been demonstrated to induce the expression of c-myc in a time-
dependent manner (Liu and Teng, 1994). In general, exposure of cells to estrogen in vitro
has not been demonstrated to be mitogenic in isolated uterine cell cultures (Tomooka et al.,
1986), nor to induce c-myc mRNA in primary endometrial epithelial cells (Jouvenot et al..
1990). Withdrawal of cells from the cell cycle by removal of growth factors leads to
down-regulation of c-myc expression and may be a requirement for growth arrest (Waters
et al., 1991). Furthermore, deregulated c-myc expression can induce apoptosis in
fibroblasts deprived of growth factors (Evan et al., 1992), suggesting that this protein may
serve to integrate the effects of different signaling pathways for cellular growth.
Endometriosis is characterized by the extrauterine proliferation of endometrial
tissue. Characterization of the effects of TCDD and B(a)P on proliferation of endometrial
cell cultures would provide a better understanding of the potential contribution of these
xenobiotics to the etiology of uterine disease. The present study indicates that B(a)P, but
not TCDD, was able to significantly decrease the proliferative ability of RL95-2 cells in
culture under both serum-free and complete growth media conditions. In fact B(a)P
treatment resulted in virtual arrest of the growth of RL95-2 cells as seen in the plateau in
cell numbers after B(a)P administration. In contrast, neither TCDD nor B(a)P were able to
alter the expression of the steady state levels of mRNA for c-myc. Therefore the inhibition
of cellular growth mediated by B(a)P does not appear to have a direct causal relationship
with c-myc mRNA levels in this cell line.
In summary, B(a)P, but not TCDD, is able to significantly decrease the expression
of immunoreactive EGF receptor protein in RL95-2 cell cultures, and there does not appear
to be a direct relationship with the ability of these agents to induce the expression of
CYPlAl and IBl, members of the cytochrome P450 family. It is possible that reactive
metabolites of B(a)P have a role to play in the process of EGF receptor down-modulation.
These data correlate with the etiological association of TCDD with enhanced and B(a)P
with decreased incidences of endometriotic lesions, respectively, insofar as previously
published data has shown no significant differences in EGF receptor expression between
normal and ectopic endometrium (Huang and Yeh, 1994; Prentice et al., 1992).
Furthermore, B(a)P, but not TCDD, is able to inhibit the cellular prohferation of RL95-2
cultures without a concomitant change in the expression of c-myc mRNA levels. Since c-
myc expression may not be directly related to the proliferative phenotype, but indirectly
involved in the process, a direct correlation may not be observable with c-myc mRNA
TCDD(nM) 0.1 1 10 50
B(a)P (10 ^iM)
Figure 3- 1 . Northern blot analysis of the effect of TCDD and B(a)P on the
CYPl Aland CYPIBI expression in RL95-2 cells. Total RNA was
denatured, blotted, and hybridized with 32P-iabeled cDNA probes for
CYPIAl and CYPIBI. (A) Dose-dependent induction of CYPl A 1 and
CYPIBI by TCDD upon a 48 hr exposure to TCDD. (B) Time-dependent
induction of CYPIAl mRNA after exposure to 10 |xM B(a)P.
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Figure 3-2. Graphical representation of the specific binding of [^^Sjj.gQp ^^
RL95-2 cells after treatment with 0-100 nM TCDD. Cells were incubated with
TCDD in the presence of 10% FBS over a 96 hr period. Cellular protein, 50 ^g
was incubated with 400pg [usjj.egf in the presence and absence of excess
(100 ng) unlabeled EOF. Data represent the mean ± SEM of at least three
separate determinations .
InM 10nM 1 jiM 20nM
Figure 3-3. Effect of TCDD and B(a)P on EGFR immunoreactive protein levels
in RL95-2 treated cultures. Actively growing cultures were treated with TCDD
or B(a)P for 48 hr and total cell lysate run on 7.5% SDS-polyacrylamide gels and
transferred onto nitrocellulose and probed with sheep anti-human EOF receptor
antiserum. (A) Western blots of representative experiments. (B) Quantitation of
changes in the level of EOF receptor protein in cultures treated with TCDD and
B(a)P. Data are the mean + SE for three experiments. * P < 0.001 compared to
E3 TCDD (10 nM)
B(a)P (10 \M.)
100- _ -r
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Figure 3-4. Quantitation of the effect of TCDD and B(a)P exposure on the
proliferation of cultures of RL95-2 cells in serum-free media. Actively
growing cultures were put into serum-free media in the presence of 0.1%
(v/v) DMSO, 10 nM TCDD, or 10 ^M B(a)P as described in Materials and
Methods. After designated times points cells were trypsinized and counted
under a light microscope. Points represent mean ± SEM from three separate
experiments. * P < 0.01 compared to control.
El TCDD (10 nM)
B(a)P (10 mM)
- - - ■- »- »
Figure 3-5. Quantitation of the effect of TCDD and B(a)P exposure on the
proUferation of cultures of RL95-2 cells in complete media. Actively growing
cultures were put into complete media in the presence of 0.1 % DMSO, 10 nM
TCDD, or 10 |iM B(a)P as described in Materials and Methods. After
designated times points cells were trypsinized and counted under a light
microscope. Points represent mean + SEM from three separate experiments.
* P < 0.01 compared to control.
B(a)P (10 ^iM)
TCDD (10 nM)
Figure 3-6. Representative Northern blot analysis of c-myc mRNA. Cells were
treated with TCDD or B(a)P up to a 48 hr time point and total RNA was isolated,
denatured, blotted and hybridized with -^^P-labeled cDNA probe for c-myc as
described in Materials and Methods. Representative Northern blot for (A) 10 |j.M
B(a)P exposure and (B)10 nM TCDD treatment of RL95-2 cultures.
EFFECTS OF TCDD AND B(a)P ON CELLULAR INVASIVENESS AND THE
EXPRESSION OF uPA AND TIMPs IN A HUMAN ENDOMETRIAL CELL LINE
In endometriosis, uterine endometrial tissue is thought to mislocate to and invade
extrauterine sites as a result of retrograde menstruation (Sampson, 1927). The growth
regulation of endometriotic tissue, however, is poorly understood since a majority of
women of reproductive age women exhibit retrograde flow, yet only around 10% of them
will manifest symptoms of the disease, (Olive and Schwartz, 1993). This suggests that
those women who develop endometriosis have endometriotic tissue which is more prone
to implant and invade the peritoneum, possibly through the action of directed and localized
extracellular proteolysis, which may involve plasminogen activators (PA) (Fernandez-
Shaw et al., 1995). Alternatively, endometriosis is also associated with the presence of
adhesions in the peritoneal cavity which has led to the suggestion that patients with
endometriosis might have a deficiency in PA activity in the peritoneal fluid, resulting in
more permanent adhesions because of reduced fibrin clearance (Malick, 1982).
The process of cellular implantation is highly regulated because dysregulation may
have profoundly undesirable consequences, as manifested in tumor metastasis. Figure 4-1
presents a simplified schematic of potential actors in the process of peritoneal implantation
based on the Sampson hypothesis (Edwards et al 1996; Stetler-Stevenson et al 1993).
Urokinase PA (uPA), a serine protease, may play a role in cellular invasion by directly
acting to degrade extra-cellular matrix (ECM) proteins such as fibronectin (Quigley et al.,
1987). In addition, uPA can also act through its ability to cleave plasminogen to plasmin,
a protease which is capable of cleaving other ECM proteins as well as activating members
of the family of enzymes known as matrix metalloproteinases (MMPs) (see Mayer 1990;
Mignatti and Rifkin, 1993 for reviews).
MMPs are a family of enzymes involved in ECM turnover and tissue remodeling
(Stetler-Stevenson et al., 1993). Their activity can in turn be modulated by interaction
with specific proteinase inhibitors known as tissue inhibitors of metalloproteinases
(TIMPs), of which four have now been characterized (Boone et al., 1990; Docherty et al.,
1985; Greene et al., 1996; Hammani et al., 1996; Silbiger et al., 1994; Stetler-Stevenson
et al., 1990). In the case of MMPs, active enzymes are inhibited by interaction with
TIMPs which form tight-binding 1:1 complexes with the MMP enzyme active sites
(Stetler-Stevenson et al., 1993). An imbalance between proteinases and their activators
and inhibitors can be imphcated in a number of pathological states including tumor
invasion (Liotta and Stetler-Stevenson 1991).
The goal of this study was to determine whether TCDD and B(a)P could alter the
invasive properties of endometrial cells as a potential etiological factor in uterine disease.
TCDD has recently been demonstrated to induce the expression of uPA in human
keratinocyte cell lines (Gaido and Maness, 1994, 1995). Our study thus evaluated the
effect of TCDD and B(a)P on the ability of RL95-2 cells to invade matrigel membranes.
The effect of these xenobiotics on the levels of uPA mRNA was also determined along
with measurement of potential changes in the fibrinolytic activity of endometrial cells
exposed to these agents. Finally, changes in the mRNA levels for TIMP 1 and 2 were also
examined in order to determine whether these xenobiotic agents are able to up- or down-
modulate other components of the tissue remodeling network.
Evaluation of the Effect of TCDD and Bfa)P on RL95-2 Cellular Attachment and
The ability of endometrial cells to invade tissue at extrauterine sites is one of the
requirements of the Sampson hypothesis (Sampson 1927). The ability of RL95-2
endometrial cultures to invade matrigel membranes after TCDD and B(a)P exposure was
evaluated utilizing a modified Boy den Chamber apparatus (Figure 4-2). The results are
represented graphically in Figure 4-3 and indicate that 10 |iM B(a)P, but not 10 nM
TCDD, was able to significantly inhibit the invasion of matrigel membranes as compared
to control cultures (p < 0.001). The numbers of invasive cells was decreased in
comparison to the control by up to 95% following a 48 hr exposure of cultures to 10 |xM
B(a)P (Figure 4-3). Cellular invasion is a multi-faceted process involving cellular
attachment and chemotaxis, as well as the degradation of basement membrane
In order to ascertain whether or not the observed alteration in cellular invasiveness
of B(a)P treated RL95-2 cultures was associated with cellular attachment, cells were
allowed to attach to matrigel membranes for up to 24 hr. The numbers of attached cells
were evaluated using a light microscope after nuclear staining. The results of a 2 hr
attachment protocol are shown in Figure 4-4 A & B. Forty-eight hr exposure to 10 ^M
B(a)P resulted in a significantly decreased ability of RL95-2 cells to attach to a matrigel
membrane compared to DMSO treated controls (p < 0.001). This result was reproducible
even for attachment periods of up to 24 hr (data not shown). It should be noted that the
cells used in the attachment and invasion assays exhibited greater than 95% viability as
determined by tryphan blue dye exclusion.
Effect of TCDD andBfalP on uPA mRNA Steady State Levels and Plasminogen Activity
in RL95-2 Cells
Northern blot analysis of the 2.8 kb mRNA transcript for uPA in RL95-2 cultures
indicated that a high level of constitutive expression exists in control cells. Exposure of
cultures to 10 nM TCDD, but not 10 ^M B(a)P, resulted in a 2-fold, time-dependent
induction of uPA mRNA steady state levels (Figure 4-5A). The observed increase in
steady state message for uPA mRNA was significant by 36 hr and remained elevated for
the duration of the 48 hr assay period (Figure 4-6B). Furthermore, this increase was also
dose-dependent, with significant induction not being observed below the 10 nM TCDD
exposure level (Figure 4-6A & B). In contrast, neither 1 |a,M nor 10 |iM B(a)P exhibited
any significant alteration in uPA mRNA levels after a 48 hr exposure of RL95-2 cells
We next chose to investigate whether the level of fibrinolytic activity in the
conditioned media from cultures exposed to TCDD and B(a)P was subsequently altered.
Fibrin zymography is a classic assay for biological activity of uPA (Figure 4-7 A) which
relies upon the ability of PA within the lanes on an SDS-PA gel to act upon plasminogen to
generate active plasmin and hence produce fibrin degradation products which show up as
lytic zones on Coomassie or amido-black stained gels (Granelli-Piperno and Reich, 1978).
Analysis of the conditioned media from TCDD and B(a)P treated cultures, showed the
presence of a single 54 kDa lytic band after staining. Evaluation of the relative sizes of the
lytic bands generated by the conditioned media from treated cultures was performed by
densitometric scanning. The analysis indicated that neither 10-100 nM TCDD, nor 10 |iM
B(a)P significantly altered fibrinolytic activity in conditioned media from cultures of RL95-
2 cells compared to 0.1% (v/v) DMSO controls (Figure 4-7B).
Effects of TCDD and BCalP on TIMP mRNA Expression in RL95-2 Cells
We next evaluated the steady state mRNA levels for the TIMPs because of their
role in the regulation of proteases important in the degradation of matrix membrane
proteins. Northern blot analysis was performed after 48 hr exposure of RL95-2 cultures
to 10 nM TCDD and 10 |jM B(a)P. Both TCDD and B(a)P treatments significantly
increased the steady state levels of the 1 kb mRNA transcript for TIMP-l relative to DMSO
treated controls (p < 0.05); however, B(a)P was able to induce relatively higher steady
state levels of TIMP-l than was TCDD (Figure 4-8 A & B). In contrast, a time course
analysis of TIMP-2 mRNA levels after treatment of cultures with 10 nM TCDD did not
demonstrate any significant change in the levels of either the 1.2 or 3.5 kb mRNA
transcripts (Figure 4-9). Thus there appears to be a selective increase in the expression of
steady state levels of TIMP-l mRNA as opposed to TIMP-2 by TCDD and B(a)P
exposure in the RL95-2 cell line.
Rier et al (1993) demonstrated that the incidence and severity of endometriotic
lesions in female rhesus monkeys was associated with their dietary exposure to TCDD in a
dose-related manner. Study of a surgically-induced model of endometriosis in rodents
similarly showed that the administration of TCDD and the pesticide methoxychlor (MTX)
significantly promoted the growth of endometriotic sites (Cummings and Metcalfe, 1995;
Cummings et al, 1996). In this regard, the physiological and molecular mechanisms
whereby endometriotic tissue develops and persists outside of the uterine cavity and
musculature are not well understood. On the basis of observed alterations in cellular and
humoral immune function in endometriosis patients, it has been hypothesized that
endometriosis may be the result of a decreased immune surveillance, recognition and
destruction of misplaced endometrial tissue (Dmowski et al., 1994). Results of our
investigation into the potential alteration in immunological components by TCDD will be
examined in Chapter five. The present chapter examined the data which relates to the
potential role of TCDD and B(a)P in the alteration of factors involved in endometiial cell
invasion and their potential contiibution to the promotion of endometriotic lesions.
The data from the present study as presented in Figure 4-3 demonstrate that B(a)P
produced a significant decrease in the ceU invasive activity of the RL95-2 endometrial cell
line. In contrast, TCDD exposure did not significantly alter the ability of the RL95-2 cells
to invade matrigel membranes. Our data with B(a)P is consistent with epidemiological
findings that cigarette smoking is correlated with significantly reduced incidence of the i
disease (Cramer et al., 1986; Mattorras et al., 1995). The fact that TCDD did not
significantiy alter the cell invasive activity of the exposed cultures impUes that this agent [
may not act to promote endometriosis by enhancing the implantation of uterine cells, but ;
may perhaps act at a later phase of growth and proliferation. Alternatively, the fact that the |
RL95-2 ceU hne is derived from a carcinoma may mean that the cell line already exhibits a '
maximal invasive phenotype. Consequently, it may be difficult to observe increases in the
invasive ability of these cells after exposure to TCDD.
Several factors should be considered in the analysis of the ability of B(a)P to
decrease overall cellular invasiveness as determined by this assay. These factors include
the requirement that the endometrial cells first attach to the matrigel membrane prior to
invasion. The possibility exists that B(a)P acts at the level of cellular attachment through a
change in the expression of ceU adhesion molecules. In this regard, we evaluated the
ability of RL95-2 cells tireated with TCDD and B(a)P to adhere to matrigel membranes.
The degree of attachment produced by B(a)P-treated cultures was comparable to the degree
of overall invasion, being significantly lower than that of TCDD or control cultures. The [
B(a)P levels used to treat the RL95-2 cultures are on the high end of that which would '
present in a heavy smoker. Furthermore, only viable cells, as determined by tryphan blue
exclusion were utilized in the invasion and attachment assays, therefore the results
observed in these experiments are most likely not the result of B(a)P cytotoxicity.
These results are consistent with the observation that primary cells from
endometriotic biopsies, but not normal endometrium, have been reported to exhibit the loss
of E-cadherin expression (Gaetje et al., 1997), while a lack of p3 integrin expression was
found to be closely correlated with a diagnosis for endometriosis (Lessey et al., 1994). In
this regard, E-cadherin is regarded as an invasion suppressor, cell adhesion molecule.
Secondly, the previous chapter presents evidence that B(a)P produced a state of growth
arrest in these endometrial cell cultures under serum-free conditions, and markedly slowed
their overall rate of prohferation in complete media. The invasion and attachment assays
were performed after treatment of RL95-2 cultures in complete media. A reduced rate of
proliferation produced by B(a)P could affect the ability of the cells to invade basement
membrane if growth and proliferation are required for the elaboration of factors involved in
the attachment/invasion process.
Based on the simplified schematic of the process of cellular invasion (Figure 4-1),
we next evaluated the potential of TCDD and B(a)P to alter the expression of specific
proteases and their inhibitors which could account mechanistically for the contributions of
these environmental agents to the etiology of endometriosis. Urokinase plasminogen
activator (uPA) is a serine protease which catalyses the conversion of plasminogen to
plasmin, another protease which is itself able to play a role in an array of processes such as
tissue growth and remodeling, tumor invasion and metastasis (Mayer, 1990). The abihty
of uPA to generate plasmin allows for the activation of members of the matrix
metalloprotease family of enzymes capable of degrading extracellular matrix proteins like
collagen and fibronectin (Stetler-Stevenson et al., 1993).
Our data provide evidence that TCDD, but not B(a)P, is able to significantly
increase the expression of steady state mRNA levels of uPA in a time- and dose-dependent
manner. Furthermore, this induction of uPA steady state mRNA levels appeared to be the
consequence of post-transcriptional processes as shown by the ability of CHX to produce
a superinduction of the mRNA message. In this regard, TCDD may be altering the
expression of protein(s) which are involved in the regulation of steady state uPA mRNA
levels. For example, the decreased expression of a protein involved in degradation of uPA
mRNA would result in an increased half-life for the uPA message, and could account for
the observed effect of TCDD on uPA mRNA levels.
We next chose to evaluate whether this observable increase in mRNA was
translated into enhanced fibrinolytic activity present in the conditioned media from
endometrial cultures exposed to TCDD and B(a)P. Our experiments did not demonstrate
any significant increase in the fibrinolytic activity of conditioned medium from either
TCDD or B(a)P treated cultures. These observations led to the conclusion that if any
differences existed, they may be present in the endometriotic tissues themselves. The fact
that no increases in fibrinolytic activity were observed for TCDD treated cultures although
there was a significant increase in steady state uPA mRNA expression could be the result
of compartmentalization. Studies of cells in culture have shown that uPA can be
compartmentalized, i.e bound to cell-surface receptors at focal contact points and remains
active when bound to its membrane localized receptor. Hence increases in uPA activity in
conditioned media might not be readily observed.
These data raise a number of interesting points of discussion. First, the ability of
TCDD to increase the steady state level of uPA mRNA has previously been demonstrated
in human keratinocytes, which further analysis showed to be the result of post-
transcriptional regulation via an increase in the half hfe of the uPA message (Gaido and
Maness, 1995). Immunohistochemical analysis of the levels of PA in the endometrium of
women with endometriosis showed a variation in the levels in normal endometrium
throughout the menstrual cycle, whereas endometriotic tissue maintained a consistently
high level of immunoreactive PA (Fernandez-Shaw et al., 1995). These results indicate a
potentially more invasive nature of the endometriotic implants.
Alternatively, Malick (1982) developed the hypothesis that changes in fibrinolytic
activity could contribute to the development of endometriosis. MaUck suggested that
decreased peritoneal fibrinolytic activity could be responsible for the adhesions seen in the
disease because of a decreased capacity to lyse fibrin deposits which develop secondary to
peritoneal injury. Human endometrial cells have been shown to release two major forms
of PA, tissue-type (tPA) and urokinase (uPA), whose expression and release are regulated
by progesterone, estrogen and EGF (Miyauchi et al., 1995a, b). The fibrin gel
zymography assay works equally well for the evaluation of either tPA or uPA fibrinolytic
activity (Granelli-Pipemo and Reich, 1978). In our study, the Ij^ic activity generated by
the conditioned medium of RL95-2 endometrial cultures is centered around a 54 kDa band
which corresponds to the molecular weight of uPA, rather than tPA which is a 70 kDa
protein. The fact that our data show no increase in the actual fibrinolytic activity of the
conditioned medium is supported by other workers who have failed to find any significant
differences in the fibrinolytic activity of peritoneal fluid from women with endometriosis
and/or pelvic adhesive disease compared to control patients (Batzofm et al., 1985;
Dunselman et al., 1988).
The present study also examined the steady state level of mRNA expression of two
members of the family of tissue inhibitors of matrix metalloproteases (TIMPs) after
treatment of endometrial cultures with TCDD and B(a)P. TIMP-1 mRNA levels are
increased by both TCDD and B(a)P exposure, with a greater level of induction by B(a)P
compared to TCDD. In comparison, neither TCDD nor B(a)P significantly altered the
levels of the two TIMP-2 mRNA transcripts. TIMPs belong to a family of proteins which
inhibit coUagenases and gelatinases, and an imbalance between proteinases and their
activators and inhibitors has been implicated in a number of pathological states including
tumor invasion, fibrosis and arthritis (Stetler-Stevenson et al., 1993). Manipulation of the
balance between MMPs and TIMPs can induce or suppress cellular invasion, as
demonstrated by the ability of the overexpression of TIMP-2 in human melanoma A2058
cells to modulate not only proteolysis of the extracellular matrix, but also the adhesive and
spreading properties of the cells (Ray and Stetler-Stevenson, 1995).
The lack of change in the mRNA levels for TIMP-2 in our RL95-2 cells may not be
at all surprising based on the recent characterization of the TIMP-2. This analysis has
shown that TIMP-2 has several features observed in housekeeping genes, with mRNAs
transcripts having longer half lives than that of p-actin (Hammani et al., 1996). This is in
contrast to TIMP-1 and TIMP-3 which exhibit highly inducible levels of mRNA
expression. The expression of TIMP-2 can be characterized as largely constitutive, in
contrast to TIMP-1 and TIMP-3 both of which are highly inducible at the transcriptional
level in response to phorbol esters and serum growth factors (Edwards et al., 1996). Thus
TIMP-2 may play a major role in providing a stable basal level of inhibitory activity in
tissues (Hammani et al., 1996). The abihty of both TCDD and B(a)P to induce TIMP-1
mRNA levels would lend support to a reduced invasiveness for these cells, but such a
phenomenon is observed only for B(a)P treated cultures. Cellular invasion is a multistep
process, involving the net co-ordinated interaction of a number of genes and gene
products. The increased TIMP-1 mRNA levels observed after TCDD and B(a)P treatment
in this cell line may, therefore, not be readily interpretable on their own, as indicators of an
alteration in overall cellular invasiveness.
In summary, B(a)P, but not TCDD, was shown to inhibit the abihty of RL95-2
endometrial cultures to attach to and traverse matrigel membranes. TCDD, but not B(a)P,
significantly increased the steady state levels of uPA mRNA, yet neither TCDD nor B(a)P
altered the fibrinolytic activity of the conditioned medium from treated cultures. Finally,
both TCDD and B(a)P enhanced the level of expression of TIMP-1 mRNA, but had no
effect on TIMP-2 expression. Further work needs to be carried out in order to better
characterize the overall effect of these environmental agents on cellular invasion and tissue
remodeling factors and so elucidate their full potential role in uterine pathologies.
ENDOMETRIAL CET J,
Figure 4-1. Illustration outlining the potential mechanism of invasion and
implantation of endometrial cells.
> * . f
Figure 4-2. Diagram of a modified Boyden Chamber apparatus.
(A) Cross-sectional view and (B) View fron above.
■ BaP (10 |iM)
□ TCDD (10 nM)
Figure 4-3. Quantitation of the effect of 10 nM TCDD and 10 i^M B(a)P
pre-treatment on the ability of RL95-2 cells to invade Matrigel membranes.
Approximately 2 x lO'* cells were aliquoted into the bottom wells of the
apparatus and allowed to attach to a 8 micron Matrigel-coated membrane for 90
min after inversion. Upper wells were loaded with complete media and cells
allowed to migrate for 36 hr. Cells which migrated through the membrane
were stained with Leukostat and counted under a light microscope. Data are
expressed as the mean ± SEM of the number of migrating cells from three
separate experiments. * P < 0.001 as compared to 0.1% DMSO controls.
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■ 10 |iM BaP
S 10 nM TCDD
Figure 4-4. Quantitation of the effect of 10 nM TCDD and 10 jiM B(a)P
pre-treatment on the ability of RL95-2 cells to attach to Matrigel membranes.
Approximately 5x10^ cells were aliquoted into the bottom wells of the
apparatus and allowed to attach to a 8 micron Matrigel-coated membrane for 2
hr after inversion. Cells which attached to the membrane were stained with
Leukostat and counted under a light microscope. (A) Scanned image of
treated RL95-2 cell attached to matrigel membranes. (B) Data expressed as
the mean ± SEM of the number of attached cells from two separate
experiments. * P < 0.001 as compared to 0.1% DMSO controls.
6h 12h 24h 48h
TCDD (10 nM) - + - + - + - +
B(a)P (10 |iM)
• igl^ Jlll~
Figure 4-5. Northern blot analysis of uPA mRNA. Cells were exposed to TCDD or
B(a)P and total RNA isolated, denatured, blotted and hybridized with 32P-labeled
cDNA probes as described in Materials and Methods. (A) Representative blot of the
time-dependence of the induction of uPA with 10 nM TCDD treatment. (B) Blot
of the effect of B(a)P exposure on uPA mRNA levels in RL95-2 cells for 48 hr.
TCDD(nM) 0.1 1 10
TCDD (10 nM)
TCDD (1 nM)
TCDD (0.1 nM)
Figure 4-6. Dose-dependent effect of TCDD on the expression of uPA
mRNA in RL95-2 cells. (A) Northern blot analysis of total RNA after a 48
hr exposure to varying concentrations of TCDD. Analysis performed as
described in Materials and Methods. (B) Graphical representation of the
dose-dependent effect of TCDD on uPA mRNA levels. Data represent the
mean scan intensity ± SEM of at least three experiments. * P < 0.05.
uPA (IN GEL LANES)
Figure 4-7. Evaluation of the fibrinolytic activity of the conditioned media from
B(a)P and TCDD treated RL95-2 cultores. (A) An outline of the methodology
mvolved m the fibrmolytic analytical technique. (B) Representative photograph
of the lytic bands produced by the plasminogen activity of the conditioned media
from the treated cultures.
# # #
C ^ *■- <^v
m TCDD (10 nM)
a B(a)P (1 ^iM)
13 B(a)P (10 ^iM)
Figure 4-8. Northern analysis of TIMP-1 mRNA. RL95-2 cultures were exposed to
TCDD, B(a)P or 0.1% DMSO for 48 hr after which total RNA was isolated,
denatured, blotted and hybridized to 32P-labeled cDNA probes as described under
Materials and Methods. (A) Representative Northern blot probed with 32p-TIMP-l.
(B) Graphical representation of the scan intensity from treated cultures. Bars represent
the mean ± SEM of at least three experiments. * P < 0.05
TCDD (10 nM)
\'' * -^ ^"'"^ ^^^'•
B(a)P (10 M,M) ^.
Figure 4-9. Northern analysis of TIMP-2 mRNA. RL92-2 cultures were exposed
to (A) 10 nM TCDD, (B) 10 ^M B(a)P, or 0. 1% DMSO for 48 hr and total RNA
isolated, denatured, blotted and hybridized to 32p-iabeled cDNA probes as
described under Materials and Methods. (A) and (B) are representative Northern
blot probed with 32P-TIMP-2 generated from cultures treated as indicated Blots
show the presence of both the 1.2 and 3.5 kb mRNA transcripts for TIMP-2.
EFFECTS OF TCDD ON IL-lp AND TNFa IN A HUMAN ENDOMETRIAL CELL
Normal endometrium undergoes predictable biochemical and histological changes in
response to hormones throughout the menstrual cycle. Cytokines including IL-1 and
TNFa are produced by cell populations within the uterine environment and may participate
in growth and differentiation of the endometrium (Frank et al., 1995; Laird et al., 1996;
Roby and Hunt, 1994; Simon et al., 1993). IL-1 and TNFa are among the pleiotropic
growth factors which may act as local mediators of cellular communication in the uterine
cavity (Hunt et al., 1992), with the IL-1 system having recently been demonstrated to play
a role in the process of embryonic implantation (Simon et al., 1994). Dysregulation of the
expression of these cytokines could potentially result in pathological disorders of the
endometrium and uterine cavity and musculatare.
The Sampson hypothesis postulates that endometriosis is a consequence of the
implantation and growth of desquamated endometrial cells and fragments at extrauterine
sites, yet this model does not take into account the prevalence of retrograde menstruation
within the female population (Halme et al., 1984). An altemative theory is based on
evidence that alterations in the normal immune system regulation may facilitate the
implantation of endometrial fragments, thereby contributing to growth, and disease
progression (Dmowski et al., 1994; Gleicher and Pratt, 1993; Rier et al., 1995). Evidence
in support of this immunologic hypothesis comes from data showing evidence of elevated
levels of inflammatory ceU products such as IL-1, IL-5, IL-6, IL-8, IL-10, TNF-a and
prostaglandins in peritoneal fluid as well as macrophage and macrophage-conditioned
media from endometriosis patients (Keenan et al., 1995; Koyama et al., 1993; Mori et al.,
1991; Rana et al., 1996; Taketani et al., 1992). In this regard, danazol, a mainstay of the
medical management of endometriosis, is able to suppress the production of IL-1 and TNF
by human monocytes (Mori et al., 1990). Further evidence for a role for immunological
factors in the maintenance of endometriotic implants is based on the ability of macrophages
to secrete cytokines capable of influencing endometrial growth, as well as the observation
that peritoneal fluid from women with endometriosis increases endometrial cell proliferation
(Ramey and Archer, 1993). Alterations in cell-mediated and humoral immunity have also
been noted in the disease (Dmowski et al., 1994; Gleicher, 1994).
TCDD is a well characterized immunotoxicant (Holsapple et al., 1996; Kerkvliet,
1995; Masten and Shiverick, 1995). TCDD has previously been shown to induce the
expression of both IL-lp and TNFa in vivo (Fan et al., 1997) and in vitro in human
keratinocytes (Sutter et al., 1991) and in MCF-7 breast cancer cells (Vogel and Abel,
1995). The ability of these compounds to alter cytokine expression could facilitate a role in
the pathogenesis of uterine disease. The objective of this study was to evaluate the effects
of TCDD on the expression of IL-1 (3 and TNFa in a human endometrial cell culture model.
The study examined the dose and time dependence of TCDD exposure with changes in
cytokine mRNA levels.
Effects of TCDD on IL-lg and TNF-a mRNA levels in RL95-2 Cells
TCDD exposure significantly increased the steady state level of mRNA for the IL-
1(3 transcript relative to controls as evaluated by Northern blot analysis (Figure 5-1). Data
show that 10 nM TCDD exposure of RL95-2 cultures resulted in a time-dependent
induction of the expression of IL-1 (3 mRNA which is observed as early as 6 hr after
treatment. Maximal induction of about 5-fold occurred by 36 hr after TCDD treatment.
with levels remaining elevated above controls for the duration of the 48 hr assay period
(Figure 5-2). Not only was the induction of IL-lp mRNA time-dependent, but it was also
observed to be dose-dependent (Figure 5-lB). A representative Northern blot performed
after a 24 hr treatment with concentrations of TCDD from 0.1-10 nM TCDD indicates that
IL-lp mRNA is induced by TCDD exposures as low as 0. 1 nM and is maximal by 10 nM
TCDD. In comparison, RL95-2 exposure to TCDD led to a significant (p < 0.05), time-
dependent induction of TNF-a mRNA steady state levels by 36 hr (Figure 5-3). Levels
plateaued at this 36 hr time point and remained elevated relative to controls for the
remainder of the 48 hr assay period (Figure 5-3B). Hence TCDD treatment resulted in
significantly increased levels of mRNA for both IL-ip and TNF-a. However, the time
course and relative levels of induction varied between both messages. IL-ip mRNA was
significantly increased at an earlier time point as well as showing higher levels of induction
at maximal levels relative to TNF-a (Figure 5-2 and 5-3). It warrants note that the late
induction of TNF-a showed a time-dependency similar to that exhibited for uPA induction
(Figure 4-5 and 5-3). |
Effect of TCDD on the Rate of Transcription of CYP1 AL CYP1B1. uPA. and n.-1J ^
mRNA in RL95-2 Cells
Nuclear runoff analyses were performed in order to determine whether the observed
changes in uPA and IL-lp mRNA levels were the result of increased transcriptional
activity. Nuclei were isolated from RL95-2 cells treated with 0.1% DMSO and 10 nM
TCDD and analyzed for the levels of nascent mRNA transcripts. Data in Figure 5-4
demonstrate that CYPlAl transcription is significantly increased 5-fold by TCDD, whereas
no effect was observed on CYPIB 1, uPA or IL-lp transcripts.
Furthermore, we also utilized the protein synthesis inhibitor cycloheximide (CHX)
to investigate whether the induction of uPA, TNF-a and IL-ip mRNA was dependent upon
the synthesis of other proteins. As shown in Figure 5-5, the exposure of cultures of RL95-
2 cells to CHX in the presence or absence of TCDD led to the superinduction of uPA,
TNF-a and IL-lp mRNA levels, implying that the levels of these mRNA are tightly
regulated and dependent upon de novo protein synthesis. Gaido et al (1995) has recentiy
shown that TCDD does in fact regulate the level of uPA mRNA in a human keratinocyte
cell line via a post transcriptional mechanism. Thus evidence indicates that TCDD is not
acting to increase the steady state levels of mRNAs for IL-lp, TNF-a and uPA by directly
altering the rate of mRNA transcription.
The present study investigated the potential of TCDD to alter the expression of
cytokine/growth factor genes which may play a role of the etiology of endometriosis. Our
data indicate that TCDD is able to induce the expression of mRNA for both IL-lp and
TNF-a in the RL95-2 cell line. In the case of IL-ip, this induction is both time- and dose-
dependent. For TNF-a, a time-dependent increase in steady state levels of mRNA was
observed with TCDD exposure. This induction of IL-lp and TNF-a by TCDD has been
demonstrated previously in human keratinocytes (Sutter et al., 1991), a breast cancer cell
line (Vogel and Abdel, 1995) and, more recently, in rat hepatic tissue after in vivo exposure
(Fang et al., 1997). Significantly, this is the first observation of these responses in uterine
cells and may lead to a better understanding of the potential actions of TCDD and related
compounds in uterine disease etiology.
IL-ip and TNF-a are pleiotropic cytokines secreted by a variety of cell types. Their
role in mediating responses in the female reproductive system and during the course of
gestation has recently come under greater scrutiny (Simon et al., 1994; Tabibzadeh, 1991).
Many of the processes occurring during menstruation are reminiscent of the inflammatory
response in terms of cellular proliferation, ischemic necrosis, stromal granulocyte
infiltration and angiogenesis (Tabibzadeh, 1991). Both cytokines have been detected in
human and rodent uterine tissue, with their expression apparently being cyclically regulated
by estrogen and progesterone levels (Frank et al., 1995; Hunt et al., 1992; Laird et al.,
1996; Roby and Hunt, 1994). The pleiotropic nature of these polypeptides, has generated
difficulty in defining their precise roles in the uterine environment.
The immunological hypothesis of endometrial promotion, has resulted in study of
IL-ip and TNF for their potential contribution to the pathology of the disease. Increased
numbers of activated macrophages have been detected in the peritoneal and mbal fluids of
infertile women with endometriosis (Haney et al., 1983). Activated macrophages secrete
IL-lp, and increased levels of this cytokine have been found in the peritoneal fluid of
women with endometriosis above that from healthy women or those treated for the disease
(Keenan et al., 1995; Mori et al., 1991; Rana et al., 1996). Danazol, one of the mainstays
of the medical management of endometriosis, results in decreased levels of these cytokines
in the peritoneal fluid of treated subjects (Mori et al., 1990; Taketani et al., 1992).
The outcome of an inflammatory cascade could lead to the synthesis of new
connective tissue and adhesion formation as weU as increased vascularity as the result of
angiogenesis, processes which could be mediated by activated macrophages through their
elaboration of pro-inflammatory cytokines. Induction of angiogenesis, procoagulant
activity and mitogenic action on fibroblasts are among the actions of IL-lp and TNF
(Tabibzadeh, 1991). IL-1 has been shown to stimulate collagen deposition and fibrinogen
formation (Posthlewaite et al., 1984), which might account for the incidence of fibrosis and
adhesions observed in advanced stages of endometriosis. Peritoneal fiuid from
endometriotic women has been shown to have a toxic effect on mouse embryo development
(Taketani et al., 1992), an observation which has been reproduced with IL-ip (Fakih et al.,
1987), although some controversy is involved in the reproducibility of this data (Schneider
et al., 1989).
The data we have presented demonstrates a time-dependence of the induction of
messages for both IL-1 [3 and TNF, with IL-ip induction being observed by 6 hr and TNF-
a being apparent by 36 hr. This observation is interesting in light of the fact that IL-lp has
been shown to stimulate the production of TNF-a in endometrial cells and cytotrophoblasts
in culture (Knofler et al., 1997; Laird et al., 1996). It has been suggested that cytokines
are integrated into an intricate network that operates by coordinated regulation of their
expression. Our observation of the time-dependent induction of mRNA levels for IL-1(3
and TNF may be a manifestation of this phenomenon.
The fact that IL-lp and TNF are characterized primarily as immune system
modulators, supports their potential role in the pathogenesis of uterine disorders like
endometriosis and may relate to effects on cellular attachment and invasion processes. IL-
lp has recently been shown to be involved in the ability of murine embryos to be
successfully implanted (Simon et al., 1994), while the adherence of endometrial stromal
cells to mesothelial cells was significantly increased by pretreatment of mesotheUal cells
with TNF-a (Zhang et al., 1993b). The latter observation may be associated with the
abihty of TNF to alter the expression of molecules involved in adhesion interactions such
as cadherin and p-catenin (Tabibzadeh et al, 1995). The role of these pleiotropic cytokines
in the process of implantation is further supported by their ability to alter the expression of
matrix metalloproteases (MMPs), enzymes involved in the degradation of basement
membrane molecules to allow for tissue remodeling (Librach et al., 1994; Sato et al.,
The present study also examined the possibility that the induction of mRNA for
these cytokines could potentially be regulated at the transcriptional level. To this end, we
utilized the method of nuclear runoff analysis to examine the translation of nascent
transcripts from TCDD-treated cultures of RL95-2 cells. Our data indicate that while
CYPlAl steady state mRNA levels were indeed increased as a result of increased
transcription rates, IL-lp and uPA mRNA levels were not (Figure 5-4). Both IL-1 and
TNF have been reported to be regulated at both the transcriptional and post-transcriptional
level (Fenton, 1992; Tabibzadeh, 1991). Data in Figure 5-5 which show the
superinduction of IL-1[3 mRNA levels after exposure of RL95-2 cultures to CHX, a protein
synthesis inhibitor, lends support to a post-transcriptional mechanism of regulation. TNF-
a, for example, is thought to be primarily controlled at the post-transcriptional level, and
cells expressing the TNF-a message may not necessarily translate it into product (Sariban
et al., 1988). In fact, TNF-a release from cytotrophoblast cultures was demonstrated to be
independent of the induction of its mRNA levels (Knofler et al., 1997).
In summary, TCDD induced the expression of the steady state levels of mRNA for
IL-lp and TNF-a in a time-dependent manner. Analysis of the transcription of IL-1|3
demonstrated that the induction was not a result of increased transcription rates, but likely
the consequence of post-transcriptional phenomena. Thus, chronic inflammatory changes
in endometriosis may be mediated at least in part by IL-lp and TNF-a. Our data showing
increased expression of IL-ip and TNF-a mRNA after TCDD exposure opens the
possibility TCDD may also play a role not only in the invasive potential of desquamated
endometrium, but also in the infertility associated with the disease.
6h 12h 24h 36h 48h
TCDD (10 nM)
+ - + -+- + - +
0.1 1 10
Figure 5- 1 . Northern blot analysis of IL- 1 p showing the dose- and time-dependence
of its induction with TCDD. (A) Representative Northern blot showing the time-
dependence of the induction of IL-lp mRNA with 10 nM TCDD exposure. (B) A
representative Northern blot showing the dose-dependence of IL-lp mRNA induction
after a 36 hr exposure to varying concentrations of TCDD. Forty ng of total RNA
was denatured, blotted and hybridized with 32P-Iabeled cDNA as described under
Materials and Methods.
: 1 — >• — 1
10 20 30 40
Figure 5-2. Graphical analysis of the induction of IL-lp mRNA in
RL95-2 cultures treated with 10 nM TCDD (A), relative to 0.1%
(v/v) DMSO (x) controls. Data represent the mean ± SEM from five
experiments. * P< 0.001 compared to controls.
30 40 50
Figure 5-3. The effect of TCDD exposure on TNF-a mRNA expression in RL95-7
£5''^-J^^ Northern analysis of TNF-a showing a time-dependent induction with "
TCDD exposure. Total RNA. 40 ^ig, was denatured, blotted and hybridized with '^P
labeled cDNA for TNF-a as described in Materials and Methods. (B) Graphical
analysis of the induction of TNF-a mRNA with time of exposure to TCDD Values
represent the mean ± SEM of three experiments. * P < 05
Figure 5-4. Effect of TCDD on the rate of IL-lp and uPA transcription. Actively
proliferating RL95-2 cultures were treated with either 10 nM TCDD (T) or 0.1%
(v/v) DMSO (C). Nuclei were isolated after 44 hr and nuclear runoffs performed
as described under methods, p-actin was used as a loading control and CYPlAl
as a positive control for transcriptional induction by TCDD. (A) Representative
autoradiogram of a runoff analysis for transcriptional induction. (B) Results of
the desitometric analysis of the band intensities after normalization to P-actin
indicating relative changes in rates of transcription. Each bar represents the
mean ± SEM of at least three experiments for 10 nM TCDD treatment.
* P< 0.0] compared to p-actin.
CHX (5 |ig/ml)
TCDD (10 nM)
Figure 5-5. Northern analysis showing superinduction of mRNA for uPA,
TNF-a and IL-lp by cycloheximide in RL95-2 cells. Fifty |ig of total RNA
from RL95-2 cultures was isolated after 12 hr treatment with the protein
synthesis inhibitor cycloheximide (CHX), in the presence or absence of 10
nM TCDD. Bands were visualized by autoradiography after membranes were
probed with "P-labelled cDNA probes.
EXPRESSION AND PURIFICATION OF RECOMBINANT AND NATIVE
PROLACTIN-LIKE PROTEE>^S B AND C
The rodent placenta is a rich source of proteins which are structurally related to
pituitary PRL. To date, fifteen of these PRL-like proteins have been identified and shown
to exhibit a temporal and cell-specific pattern of expression. It has been suggested that
these proteins are members of a superfamily of hormones and cytokines, the helix-bundle-
peptide (HBP) hormones, with shared structaral features and mechanisms of action. The
HBP structure consists of four long a-hehces arranged in antiparallel fashion (Horseman
and Yi-Lee 1994) with family members including GH, PRL, erythropoietin and the
interleukins. Based on their homology to PRL, some of these placental proteins have been
characterized and shown to possess PRL-like bioactivity (Cohick et al., 1995; Colosi et al.,
1987a, b; Deb et al., 1991a; Robertson et al, 1982, 1994). The biological activity of a
number of these proteins, however, remains as yet undetermined.
Our laboratory had previously identified four major rat placental proteins secreted
into conditioned media from placental explant cultures. These proteins were identified by
N-terminal sequence analysis to be related to the PRL/GH family (Ogilvie et al., 1990a). A
cDNA coding for a protein virtually identical to that of Proteins 2 and 4 was later isolated
by Deb et al (1991b) and designated PLP-C based on an akeady established nomenclature
(Duckworth et al., 1986b, 1988). Our laboratory has studied several models of low birth
weight in pregnant rats and found that maternal xenobiotic exposure and protein
malnutrition, respectively, are associated with decreased placental growth, vascular
development and intrauterine growth retardation (lUGR). Moreover, growth-retarded
placentas showed decreased expression of protein and mRNA for PLP-B (ConliflFe et al.,
1995; Shiverick et al., 1991). Thus, previous evidence supports an association between
impaired placental growth and decreased expression of PLP-B and PLP-C proteins.
In an effort to identify the biological role of PLP-B and PLP-C in placental growth,
our laboratoiy initiated efforts to purify native forms of the placental protein, as well as
attempt recombinant expression of PLP-B and PLP-C proteins in both bacterial and
mammalian systems. This section of the study details the expression and purification of
recombinant PLP-C using two different bacterial constructs, as well as the mammalian
expression and purification of PLP-B. Recombinant PLP-C was further used to generate a
polyclonal antisera which was essential for immunoaffmity purification of the native protein
from conditioned media ofbasal zone explant cultures. Finally, this section also details the
use of an antipeptide antisera previously generated against the C-terminus of PLP-B
(Ogilvie et al., 1990b) for purification of native protein.
Purification of Recombinant hCAII-PLP-C
In the hCA-n fusion vector p0304 (Van Heeke et al., 1993), the DNA sequence
encoding the recombinant protein is linked to hCA-II through a cleavage recognition
sequence for enterokinase. In order to achieve the expression and subsequent release of
PLP-C from the hCAII fusion protein, it was necessary to subclone the DNA fragment
encoding PLP-C into an EcoRV site located adjacent to the enterokinase cleavage site such
that a DmI site would be present immediately adjacent to the codon specifying the first
amino acid in the mature form of PLP-C (Conliffe et al., 1994). Aliquots from different
stages in the purification of recombinant PLP-C were separated on 10% SDS-PA gels
under reducing conditions and proteins were visualized by Coomassie staining (Figure 6-
1). The predicted molecular weight of the hCAII-PLP-C fusion protein is 53,620 (hCAII,
MW 31,234; PLP-C, MW 22,386). As shown in Figure 6-1, lanes A and D, a band
corresponding to a protein of 53 kDa was purified from the bacterial extract. The linkage
of PLP-C to hCAQ allowed for rapid purification of the fusion construct utilizing a pAMBS
resin with high affinity for hCAII. The 53 kDa band comprised approximately 15% of the
Coomassie blue-stained affmity-purified protein (Figure 6-1, lane D).
Western immunoblot analysis using a PLP-C antipeptide antisera revealed a broad
band of 31-36 kDa and another band of 53 kDa in the cell lysate and pAMBS-purified
fusion protein preparations (Figure 6-2, left panel, lanes A and D.). The immunoreactive
protein of 53 kDa corresponded to the calculated molecular weight of the fusion construct
of hCAII-PLP-C and also cross reacted with hCAII antisera (Figure 6-2, right panel, lane
D). The broad 31-36 kDa immunoreactive band comprised about 75% of the purified
fusion protein and the molecular weight was confirmed by Mass Spectrometry to be 31-36
kDa. These lower molecular weight bands were initially thought to be hCAII-PLP-C
proteolytic products resulting from the action of bacterial proteases released during ceU
lysis. The addition of the protease inhibitors, phenylmetiiylsulfonyl fluoride, leupeptin,
aprotinin and EDTA during cell lysis, however, did not reduce the staining intensity of
these bands (data not shown). These bands also stained positively with a hCAII antisera
(Figure 6-2, right panel, lanes A and D) and had an N-terminal sequence, His-His-Trp-
Gly-Tyr-Gly-Lys-His-Asn-Gly, which was identical to that of hCAII. Thus, data indicate
that the immunoreactive bands of 53 kDa, as well as the 31-36 kDa were hCAII fusion
Enterokinase cleavage of the affinity-purified fusion protein produced an
immunoreactive protein band of approximately 25 kDa (Figure 6-3, lanes A and B),
corresponding to the calculated molecular weight of PLP-C. As judged by Coomassie blue
staining, the 25 kDa protein was highly enriched by affinity purification (Fig. 6-3, lane B).
Recombinant PLP-C yield varied with different preparations. Enterokinase cleavage of
PLP-C from its fusion partner produced approximately 15% recombinant PLP-C. Efforts
were made to maximize cleavage conditions, by increasing enzyme concentration and
incubation times, as well as by changing Ca2+ concentration, but there was not a significant
increase in yield of PLP-C protein.
Recombinant Expression and Purification of pET22b('+)His-PLP-C
Because of the poor yield of the original hCAII-PLP-C construct in generating
recombinant PLP-C, we undertook the construction of an alternative bacterial expression
system (Figure 6-4). Expression of recombinant PLP-C with a polyhistidine tag at its N-
terminus was chosen because this system allowed for purification using the ability of poly-
histidine to bind divalent metal ions even under denaturing conditions (Hochuli et al.,
1987). To achieve the expression and subsequent release of PLP-C protein from the
pET22b(+) expression system, it was necessary to anneal a polyhistidine primer to the 5'
end of the PLP-C cDNA from the original hCAII-PLP-C vector which included the intact
enterokinase linker region. The resulting construct was then hgated into the NdellNotl
sites of the pET22b(+) expression vector as described under Materials and Methods and as
outlined in Figure 6-4. The presence and orientation of the desired insert was determined
by restriction enzyme digestion with Ndel which would result in the release of an
approximate 700 bp fragment of the PLP-C cDNA as shown in Figure 6-5.
Aliquots from diiferent steps in the purification of recombinant PLP-C from the
pET22b(+)His-PLP-C system were separated by 10% SDS-PAGE under reducing
conditions and proteins visualized by Coomassie staining (Figure 6-6). The recombinant
protein of approximately 27 kDa is seen in lanes A , D, E, G and H of Figure 6-6A. The
figure shows steps from two different purification schemes. Lanes A-D are from the
soluble fraction of the bacterial pellet. The recovery of mostly insoluble protein (lanes E-
H) required detergent solubilization of the pellet in N-laurylsarcosine (detergent), followed
by refolding under alkaline conditions at room temperature overnight (Luck et al., 1991,
1992). The His-PLP-C protein was then isolated from the supernatant by affinity
purification on immobilized nickel cation columns. Figure 6-6B is a Coomassie stained gel
which illustrates the results of the enterokinase cleavage of the His-PLP-C protein to
generate the full length recombinant protein. Lane A represents the full length protein after
enterokinase cleavage of the His-PLP-C product (Lane B). The 25 kDa protein was
electroblotted onto PVDF membrane and amino terminal sequence analysis revealed a major
sequence of Ile-Pro-Ala-Cys-Met-Val-Glu. This sequence was identical to that of amino
acids 1-7 of PLP-C (Deb et al., 1991b, c; Ogilvie et al., 1990b). A minor species, less
than 25%, was also detected, with N-terminal sequence corresponding to residues 6-12 of
Expression of Recombinant PLP-B
The expression of reconbinant PLP-B in a mammalian system was chosen because
of the fact that the native protein exists only as a glycosylated species (Duckworth et al.,
1986b). The complete cDNA coding sequence for PLP-B was blunt-end Ugated into the
pMXSND expression vector as outlined in Figure 6-7. This vector has previously proven
useful in the generation of recombinant proteins for other members of the PRL/GH family
(Cohick et al., 1997; Deb et al., 1993; Lee and Nathans, 1988). Blunt-ended ligation of
the PLP-B cDNA was necessary because there was only a single Xhol cloning site in the
pMXSND vector with no other compatible site for 'sticky ended' ligation with the PLP-B
BamHl diagnostic restriction endonuclease digests were performed to determine the
presence of the PLP-B insert in the correct orientation. Figure 6-8 illustrates the results of
digests performed upon putative plasmids containing the PLP-B insert. Lanes 8 and 10
show results of the diagnostic digests of plasmids incorporating the pMXSND-PLP-B
insert in the incorrect and correct orientation, respectively. CHOKl cells were transfected
with the pMXSND-PLP-B plasmid and stable transfectants selected by culture in the
presence of G418. Stable clones were selected after expansion by probing for the presence
of mRNA species which hybridized to ^^P-PLP-B cDNA on Northern blot analysis (Figure
6-9). Clones 4, 5 and 7 were observed to give positive hybridization signals and were
consequently expanded by growth in large cultures with G418 and MTX selection. PLP-B
secretion was induced by the culture of stable transfectants in the presence of CdClj which
makes use of the metallothionein promoter in the pMXSND vector. Western immunoblot
analysis of concentrated conditioned media from these stably transfected cultures was
performed using an antipeptide antisera against the C-terminus of PLP-B (Ogilvie et al.,
1990b) and demonstrated the presence of immunoreactive protein of approximately 30 kDa
(Figure 6-10) which corresponded in size to native PLP-B protein (Ogilvie et al., 1990a).
Purification and Western Immunoblot Analysis of Native PLP-B and PLP-C
Native PLP-B and PLP-C were purified from the conditioned media of gestation
day 18 rat placental (basal zone) explant cultures. The utility of antisera generated against
these proteins (Ogilvie et al., 1900b; Conliffe et al., 1994) in immunoafFinity
chromatography was demonstrated. The purified native preparations of PLP-B and PLP-C
were characterized by two dimensional SDS-PAGE. As seen in Figure 6-11, PLP-B is
present as a series of 30 kDa subforms with pi ranging between 6.1-6.6, as observed by
silver staining (Figure 6- 11 A) and Western immunoblot analysis (Figure 6-llB). The anti-
PLP-C column retained species of 25 kDa and 29 kDa with pi ranging from 5.8-6.2, as
observed on silver stained two dimensional SDS-PAGE (Figure 6-12A) and confirmed by
Western immunoblot analysis (Figure 6-12B). These data are in agreement with the
previously reported size and pi characteristics of secreted PLP-B (Ogilvie et al., 1990a) and
PLP-C (Conliffe et al., 1994).
The present study describes the recombinant expression and purification of PLP-B
and PLP-C, two members of the PRL-GH family produced by the rat placenta.
Furthermore, the study also describes the purification of these native proteins from
placental explant cultures by immunoaffinity chromatography with the use of antibodies
generated against PLP-B and PLP-C. Recombinant PLP-C was initially expressed as a
fusion partner with human carbonic anhydrase (hCAII) in JM109(DE3) E. coli bacteria.
The usefulness of hCAII has been previously demonstrated as a partner in fusion protein
expression constructs with E.coli Fl-ATPase e subunit and asparagine synthethase A,
respectively, (Hinchman and Schuster, 1992; \kn Heeke et al., 1993). In the present
study, linkage of PLP-C to hCAII allowed the fusion protein to be purified to homogeneity
in a single step on a sulfonamide affinity resin. The desired 53.4 kDa fusion protein was
produced in our expression system in addition to several other hCAII fusion proteins of 31-
36 kDa. These lower molecular weight proteins may represent premature translation
termination products of PLP-C. In this regard, it is possible that chain termination might
be due to differences in codon usage between mammalian and bacterial systems.
In the expression system for the hCAII fusion protein, the recombinant PLP-C
protein is released from its fusion partner by enterokinase cleavage. Although enterokinase
digestion of hCAII-PLP-C released recombinant PLP-C, the yield was found to be highly
variable. Because the enterokinase products of the 31-36 kDa bands would not exceed 6
kDa, it is expected that they are excluded from the preparation during the centriprep
concentration step which has a 10 kDa cutoff membrane. In this regard, Hinchman and
Schuster (1992) were unable to obtain any cleaved product in the expression of asparagine
synthethase A. In contrast, release of PLP-C from the His-PLP-C construct was achieved
with a high level of consistency. This disparity in release of the recombinant protein may
be attributed to differences in enterokinase preparations or, alternatively, to folding of the
recombinant protein which might interfere with cleavage.
The difficulties encountered in the use of the hCAII fusion system for the
expression of recombinant PLP-C necessitated the development of an alternative expression
construct. The enterokinase PLP-C sequence was excised from the original hCAII-PLP-C
construct and ligated to a polyhistidine tract at the N-terminal region of PLP-C upstream of
the enterokinase region. The presence of the polyhistidine region allowed for purification
based on histidine affinity for divalent metal ions by passage over a nickel column. The
removal of the approximately 3 kDa N-terminal polyhistidine extension was effected by
incubation with enterokinase which released the recombinant PLP-C. This polyhistidine
fragment was assumed to be excluded after dialysis with a 6 kDa membrane.
The recombinant PLP-C protein isolated from the two bacterial systems had similar
molecular weight and N-terminal amino acid sequence identity with that of native placental
PLP-C (Deb et al., 1991b; Ogilvie et al., 1990b). A minor species corresponding to PLP-
C beginning at residue 7 was also detected in the expression system. Studies describing
the expression of recombinant rat placental PLP-A in a mainmalian ceU line also identified
minor species representing N-terminally truncated isoforms of PLP-A (Deb et al., 1993).
The antiserum generated against recombinant PLP-C demonstrated cross reactivity with the
25 kDa non-glycosylated and the 29 kDa glycosylated forms of PLP-C in Westem
This study also described the expression of PLP-B as a recombinant protein
utilizing a mammalian expression vector pMXSND. This vector has previously been
shown to be effective in the expression of other members of the PRL-GH family from both
rat and murine sources (Cohick et al., 1997; Colosi et al., 1988; Deb et al., 1993; Lee and
Nathans, 1988). The choice of mammalian expression was also important based on the
fact that the native protein has been detected only as a 30 kDa glycosylated species (Ogilvie
et al, 1990b; Duckworth et al., 1988). The lack of cloning sites within pMXSND
necessitated that the PLP-B cDNAbe blunt-end ligated into the vector and that transformed
colonies be screened for the presence of the PLP-B cDNA insert in the correct orientation
by restriction enzyme digestion prior to transfection into CHOKl cells. Stable transfectants
were selected after Northern blot analysis using a ^^P-labeled cDNA probe. The
conditioned medium of stably transfected clonal colonies exhibited the presence of a 30 kDa
immunoreactive band in Western immunoblot analysis corresponding in molecular weight
to native placental PLP-B protein.
This study further described the purification of native preparations of PLP-B and
PLP-C from placental explant conditioned medium in immunoaffinity chromatography
using antibodies generated against PLP-B and PLP-C. Purified native PLP-B preparations
were separated by 2D-SDS polyacrylamide gel electrophoresis and Western immunoblot
analysis demonstrated the presence of 30 kDa molecular weight species with similar
molecular weight and pi to that of the native protein (Ogilvie et al., 1990b). Similarly,
purified native PLP-C preparations showed the presence of several immunoreactive species
in Western immunoblot analysis as well as on silver stained gels. The presence of both the
25 kDa non-glycosylated and 29 kDa glycosylated forms of native placental PLP-C has
been previously characterized (Deb et al., 1991b). The other minor species in these
preparations may represent products of genes which are closely related to PLP-B and PLP-
C or, alternatively, are post-translational isoforms of these proteins.
In summary, PLP-B and PLP-C were both successfully expressed as recombinant
proteins in mammalian and bacterial systems, respectively. Placental PLP-C is expressed
as both non-glycosylated and glycosylated species. Our efforts to express PLP-C in
mammalian systems were not successful. In this regard, other researchers in the field have
similarly demonstrated an inability to recombinantly express PLP-C in a mammalian system
(M. Soares, personal communication). Antiserum was generated against PLP-C
recombinant protein and, along with an antipeptide antiserum against PLP-B, utilized in the
immunoaffinity purification of the native proteins from placental explant conditioned
medium. The availability of these protein preparations should allow for their further
Figure 6-1. SDS-PAGE analysis of the purification of hCAII-PLP-C fusion
protein. AUquots from different steps in the purification of hCAII-PLP-C fusion
protein were separated on 10% polyacrylamide gels under reducing conditions and
visualized by Coomassie brilliant blue staining. Lane A, 50 |ig cell lysate prior to
affinity purification; lane B, 50 |xg cell lysate after affinity purification; lane C, 50
|ig pAMBS column wash; lane D, 15 p.g affinity purified fusion protein; molecular
weight standards (x 10^) kDa are indicated on the left.
A B C D
Figure 6-2. Western immunoblot analysis of hCAII-PLP-C fusion protein
preparations following each purification step. Samples were separated on 10%
polyacrylamide gels under reducing conditions and analysed by immunoblotting
using antisera (1:800) raised against PLP-C oligopeptide (Fig.6-2, left panel) or
hCAII (Fig. 6-2, right panel). Lane A, 50 |a,g cell lysate prior to affinity
purification; lane B, 50 |j,g cell lysate after affinity purification; lane C, 50 |xg
pAMBS column wash; lane D, 15 |j,g affinity purified fusion protein; molecular
weight standards (x 103) ]^£)a are indicated on the left.
Figure 6-3. SDS-PAGE analysis of hCAII-PLP-C fusion protein cleaved by
enterokinase. The recombinant PLP-C was separated from hCAII by pAMBS
affinity chromatography, and then concentrated and dialysed. Samples were
separated on 10% gels under reducing conditions and stained with Coomassie
brilhant blue. Lane A, 25 |ig enterokinase-cleaved proteins; lane B, 3.5 |i.g
purified recombinant PLP-C; molecular weight standards (x 10^) kDa are indicated
on the left.
hCAII I PLP-C I
pET22b(+) His-Tag PLP-C
Figure 6-4. Schematic of the construction of a vector expressing PLP-C with an
N-terminal polyhistidine tract. The top line shows the pET22b(+) plasmid and the
hCAII-PLP-C vector being cut with restriction enzymes as indicated. The middle
lines show the three way ligation of pET22b(-i-), an oligo encoding a 9 mer
polyhistidine tract and the PLP-C cDNA fragment excised from the hCAILPLP-C
vector. The bottom line shows the structure of the pET22b(-l-)His-PLP-C vector
and the position of the enterokinase cleavage site at the junction of the histidine
tract and the PLP-C cDNA insert.
+ - + - +
Figure 6-5. Ndel diagnostic digests of plasmids from JM109 bacterial colonies
transformed witli the pET22b(+)His-PLP-C ligation mix. Photograph shows the
diagnostic cleavage of five plasmids with Ndel for the presence of the PLP-C
insert. (-) indicates an uncut plasmid, while (+) indicates the plasmid after
overnight incubation with restriction enzyme Ndel. Plasmid number 2 in the fifth
lane from the left shows the presence of the expected 700 bp PLP-C insert after
Figure 6-6. SDS-PAGE analysis of the steps in the purification of pET22b(+)His-
PLP-C protein. Aliquots were separated on 10% polyacrylamide gels under
reducing conditions and visualized by Coomassie brilhant blue staining. (A). Lane
A, 10 fig cell lysate prior to passage over the nickel affinity column; lane B, 10 |ig
cell lysate after affinity purification; lane C, 10 fig column wash; lane D, 3 [ig
affinity purified protein; Lane E, 10 |a.g of the bacterial pellet solubilized in N-
lauroylsarcosine (detergent), lane F, 10 jig solubihzed after passage over the nickel
column; lane G, 3 \ig column eluate; lane D, 3 [ig affinity purified protein after
dialysis. (B). SDS-PAGE of enterokinase cleaved pET22b(-H)His-PLP-C protein.
The protein was digested with enterokinase (Lane A) and recombinant PLP-C
separated from the His tag by dialysis (Lane B). Molecular weight standards
(xl03) are indicated on the left.
pGEM-3 PLP-B pGEM-3
I Klenow, dNTPs
Figure 6-7. Schematic of the construction of a mammalian vector expressing PLP-
B. The top line shows the pMXSND vector and the pGEM3-PLP-B plasmid being
cut with the relevant restriction enzymes as indicated. The second and third lines
show the creation of blunt-ended pMXSND and the PLP-B cDNA fragment
preparatory to Ugation after dephosphorylation of the pMXSND vector. The
bottom line shows the structure of the pMXSND-PLP-B vector.
123456789 10 11
Figure 6-8. BamHI diagnostic digests of plasmids from JM109 bacterial colonies
transformed with the pMXSND-PLP-B ligation mix. Photograph shows the
diagnostic cleavage of eleven plasmids with BamHI for the presence of the PLP-B
insert in the correct orintation. The plasmid in lane number 7 and 10 from the left
shows the presence of the PLP-B insert after BamHI digestion. Lane 10 possesses
the insert in the correct orientation as seen by the expected 1250 bp fragment as
opposed to the 750 bp fragment in lane 7.
Figure 6-9. Northern blot analysis of total RNA from clonal colonies of CHOKI
cells transfected with the pMXSND-PLP-B expression vector. RNA was
denatured, electrophoresed, transferred onto nylon membranes and hybridized with
32P-PLP-B. Colonies 4, 5 and 7 were amplified and used for the purification of
PLP-B from conditioned medium.
6 Hg 12 ng 18 ng
Figure 6-10. Western immunoblot analyses of PLP-B levels in the conditioned
media from stably transfected CHOKl cells. Samples were separated on 10%
polyacrylamide gels under reducing conditions and analysed by immunoblotting
using antisera (1:1000) raised against PLP-B oligopeptide Increasing quantities of
conditioned media from CHOKI cells stably transfected with the pMXSND-PLP-B
construct. Arrow indicates the location of the PLP-B immunoreactive band. 0,
represents conditioned medium which had passed through a 10 kDa cutoff
membrane used in the protein concentrating step. Molecular weight standards (x
103) are indicated on the left.
Figure 6-11. Two dimensional SDS-PAGE analyses of PLP-B preparations.
Native protein was immunopurified by immunoaffinity chromatography and
electrophoresed on 2D gels and analysed by silver staining for PLP-B (Figure 6-
11 A). Samples were run on duplicate gels and electroblotted onto nitrocellulose
and analysed by Western immunostaining with antisera to PLP-B (Figure 6-1 IB).
Blots were probed with an antipeptide antiserum against PLP-B at 1 : 1000 dilution,
followed by incubation with horse-radish peroxidase conjugated with anti-rabbit
IgG. Bands were visualized by incubation with 3-amino-9-ethylcarbazole in the
presence of 0.0157o hydrogen peroxide.
Figure 6-12. Two dimensional SDS-PAGE analysis of PLP-C preparations.
Native protein was purified by immunoaffinity chromatography and
electrophoresed on 2D gels and analysed by silver staining for PLP-C (Figure 6-
12A). Samples were run on duplicate gels and electroblotted onto nitrocellulose
and analysed by Western immunostaining with antisera to PLP-C (Figure 6-12B).
Blots were probed with an antiserum against recombinant PLP-C at 1:1000
dilution, followed by incubation with horse-radish peroxidase conjugated with
anti-rabbit IgG. Bands were visualized by incubation with 3-amino-9-
ethylcarbazole in the presence of 0.015% hydrogen peroxide.
CHARACTERIZATION OF NATIVE AND RECOMBINANT PROLACTIN-LIKE
PROTEINS B AND C
The recombinant proteins PLP-B and PLP-C were expressed and purified as
described in the previous chapter. In addition, native forms of the PLP-B and PLP-C
proteins were purified from the conditioned medium of basal zone explant cultures by
immunoaffinity chromatography. This chapter discusses the evaluation of the biological
activity of the purified proteins. Both the potential lactogenic and angiogenic activities have
been evaluated using standard in vitro biological assay systems.
Firstly, PRL is known to exhibit a broad range of distinct physiological actions
important in the course of reproduction (Handwerger et al., 1992). The secretion by the
placenta of high levels of placental lactogens and PRL-related proteins during pregnancy
has led to the hypothesis that these proteins are of importance in the placental-fetal growth
axis. Secondly, during the course of gestation, fetal growth and development require
enhanced placental transport and uterine blood flow which is dependent on placental
neovascularization (Reynolds et al., 1986). Two members of the placental PRL-GH
family, proliferin (PLF) and proUferin related protein (PRP), have recently been shown to
display angiogenic and angiolytic activity, respectively (Jackson et al., 1994). Our
laboratory has previously studied several animal models for low birth weight and found
that impaired placental growth and vascular development was correlated with decreased
secretion of PLP-B and PLP-C proteins by rat placental tissue (Conliffe et al., 1995;
Shiverick et al., 1991). These observations led us to further investigate the potential for
these two proteins, PLP-B and PLP-C to exhibit lactogenic and angiogenic activities.
Effects of Native PLP-B and PLP-C and Recombinant PLP-C on Nb2 Lymphoma Cell
Native PLP-B and PLP-C were evaluated for lactogenic activity utilizing a classic
cell proliferation bioassay technique (Gout et al., 1980; Tanaka et al., 1980) with Nb2
lymphoma cells. Ovine PRL was used as a positive control and produced a dose-
dependent stimulation of ^H-thymidine uptake with an EC,o of approximately 0. 1 nM.
Both native PLP-B and PLP-C exhibited some lactogenic activity but only at concentrations
above 100 ng/ml (Figure 7-1). The maximal activity of native PLP-B and PLP-C observed
was, therefore, at concentrations which were at least 3 orders of magnitude greater than that
of standard oPRL controls. The two PLP-B and PLP-C preparations were further analyzed
for the presence of PRL and PL-II by Western immunoblot analysis in order to determine
whether residual amounts of these known lactogenic placental proteins in the native PLP
preparations could account for the observed activity. In data not shown, immunoreactive
bands corresponding to the PL-II or PRL proteins were not observed, nor were these
bands observed on silver-stained 2D gels of these PLP proteins. By comparison to the
native preparation, recombinant PLP-C purified from bacterial expression systems did not
exhibit any lactogenic activity relative to the ovine PRL control in this bioassay (Figure 7-
2). Thus, evidence suggests that the lactogenic activity expressed by the native PLP-C
protein preparation may be a consequence of glycosylation which was absent from the
recombinant protein expressed in bacteria.
Effects of Placental Conditioned Medium, and Native PLP-B and PLP-C on Endothelial
Gestation day 18 placental tissue was cultured in serum-free medium for 24 hr and
the conditioned medium evaluated for the ability to stimulate the directional migration of
human retinal endothelial cells as a measure of angiogenic activity. Medium from the
cultures of placental (basal zone) tissue markedly stimulated the number of endotheUal
cells/well migrating through the collagen-coated membranes. The stimulating activity in
placental conditioned media was highly significant relative to that exhibited by the 10%
FBS positive controls (Figure 7-3), being 5-7 fold greater than that of the negative controls
(p < 0.001). In addition, the stimulatory activity of placental conditioned media appears to
be dose-related in that the response was greater with exposure to 250 [ig compared to 50
|ig of secreted protein.
In the cell migration assay, neither native PLP-B nor PLP-C preparations exhibited
any significant angiogenic activity, even at concentrations up to 5 |J.g/ml (Figure 7-4).
These data are representative of three experiments with different preparations of native
PLP-B and PLP-C. The results indicate that day 18 basal zone conditioned medium
possesses secreted, soluble angiogenic factor(s), whereas native PLP-B and PLP-C
immunopurified from this media do not exhibit any angiogenic activity as measured by
chemotactic activity towards endotheUal cells.
The secretion by the placenta of high levels of placental lactogens and PRL-related
proteins during the course of pregnancy has led to the hypothesis that they may be of
importance in the placental-fetal growth axis. Hence there is great interest in determining
whether or not these newly discovered proteins were "PRL-like" in their ability to bind the
PRL receptor or to exhibit similar bioactivities. Studies characterizing these rodent
placental proteins have demonstrated that some of them do in fact exhibit PRL-like activities
in their ability to produce lactogenic responses in rat Nb2 lymphoma cells (Gout et al.,
1980) and in mammary gland epithelial cell differentiation (Handwerger et al., 1984;,
Soares et al., 1983; Southard et al., 1986). Mouse and rat PL-I, II and Iv all exhibit such
activity, and rPL-I has been reported to be even more stimulatory than ovine PRL in the
As members of the PRL-GH family, both native PLP-B and PLP-C as weU as
recombinant PLP-C were evaluated for potential lactogenic bioactivity. The native protein
preparations exhibited some detectable activity relative to that of ovine PRL in the Nb2
lymphoma bioassay, but at concentrations which were several orders of magnitude greater
than that for the EC50 of ovine PRL. In this regard, the native preparations of PLP-B and
PLP-C were further evaluated for the presence of contaminating residual amounts of PRL
and PL-II (known lactogenic proteins) by Western immunoblot analysis and silver staining
(data not shown) of 2D gels. The results, however, were negative for the presence of
either known lactogenic protein. The presence of measurable lactogenic activity in native
PLP-C led us to express a recombinant form of the protein for further evaluation of
biological activity. When analyzed in the Nb2 lymphoma bioassay, however, recombinant
PLP-C had no mitogenic activity. Furthermore, in data not shown, neither native PLP-B
nor PLP-C were able to compete effectively for PRL binding to the long form of the PRL-
receptor, nor were recombinant forms of the two proteins able to compete for binding to the
rat PRL receptor (K. Shiverick, personal communication).
An analysis of the potential PLP-B and PLP-C three dimensional structure would
lend support to these findings. The structure of the PRL-GH family of proteins, based on
the X-ray crystallographic analysis of GH and mutagenesis studies in PRL, is thought to be
composed of an up-up-down-down, four a-helical bundle motif, linked by interconnecting
loops (Abdel-Meguid et al., 1987; de Vos et al., 1992; Goffin et al., 1992, 1993, 1994,
1996; Somers et al., 1994). As a consequence of the placental PRL-like proteins being
folded to form this four a helix type structure, the amino acid residues exposed on then-
surfaces will vary since the helical regions are not highly conserved, with the exception of
helix 4. This probably allows for variations in the properties available for interaction with
other macromolecules (Goffin et al., 1996; Southard and Talamantes, 1991), this being true
for the lactogenic as well as non-lactogenic members of the family. Mutational analysis of
the lactogenic mouse PL-II (mPL-II), along with sequence comparisons with the other
members of the placental PRL family, led to the localization of several residues essential for
maximal receptor binding and bioactivity (Davis and Linzer, 1989). These residues,
A15I9, AiglTl, Aigl75, Lysl85, Asnl95 on mPRL, map to helix 1 and 4 of the putative
four a helical folding pattern.
For the rat placental proteins of unknown biological activity, PLP-B lacks two of
the key lactogenic residues while PLP-C has none of them. The fact that recombinant PLP-
C did not exhibit the lactogenic activity observed for the native protein may also be a
consequence of the lack of glycosylation of the recombinantiy expressed bacterial protein.
Altematively, native preparations possessed some undetected soluble factor with significant
lactogenic activity. Croze et al (1990) isolated a cDNA from rat decidua identical to the
previously identified placental PLP-B cDNA. The expression of decidual PLP-B mRNA
early in gestation after implantation to midgestation prompted Croze and co-workers (1990)
to suggest that PLP-B could be a candidate gene which encodes the decidual luteotrophin of
pregnancy. This protein has been shown to specifically interact with the luteal PRL
receptor and stimulate the corpus luteum to produce progesterone (Jayatilak et al., 1989).
The low potency of lactogenic response by native PLP-B and absence of high afiinity
binding to the PRL receptor (data not shown) is evidence that PLP-B is not the decidual
Jackson et al (1994) has recently demonstrated that the midgestation mouse placenta
secretes an angiogenic activity that corresponds primarily to PLF, followed by a factor that
inhibits angiogenesis which is PRE Recombinant PLF and PRP were further found to
modulate the migration of endothelial cells in vitro and neovascularization in vivo (Jackson
et al., 1994; Volpert et al., 1996). Previous analysis of the receptor binding activity of
recombinant PLF showed a specific association with the insuhn-like growth factor
IFmannose 6-phosphate (M6P) receptor (Lee and Nathans, 1988). More recent data appear
to indicate that PLF manifests its angiogenic activity as a result of its interaction with this
receptor (Volpert et al., 1996). Data from our laboratory indicated that both the mRNA and
protein for placental PLP-B were significantly decreased in a low birth weight rat model
where reduced placental vascular development was also observed (Conliffe et al., 1995).
These observations led to the evaluation of native PLP-B and PLP-C for potential
angiogenic activity in an attempt to elucidate their role in fetal-placental development.
Our data clearly show that gestation day 18 rat placenta secretes soluble factors
which significantly stimulate endothelial ceU migration. Our finding that late gestation rat
placenta expresses predominantly stimulatory factors is in contrast with the report of
Jackson et al (1994) that conditioned medium from late gestation mouse placental cultures
was inhibitory to endothelial cell migration. The difference may be a reflection of our use
of the basal zone (junctional zone) tissue for explant culture as opposed to the whole
placenta which includes the labyrinth zone (Davies and Glasser, 1968). An additional
possibility is that endothelial cells isolated from human retina differ from bovine adrenal
capillaries in their responsiveness to chemotactic factors. In this regard, research on bovine
models shows that the fetal placental tissue produces factors which inhibit endothelial cell
migration and proliferation throughout the course of gestation (Reynolds and Redmer,
1995). Thus some evidence suggests that where these factors may function to limit
vascular development is within the maternal placental vasculature.
The present study has also found that PLP-B and PLP-C immunopurified from the
conditioned media did not show chemotactic activity towards endothelial cells. Thus it
appears that other soluble angiogenic factors are released by the rat placental explants,
which may include insuhn-like growth factors (IGF) (Grant et al., 1987, 1992), as well as
basic fibroblast growth factor (Hondermarck et al., 1990).
In summary, native PLP-B and PLP-C purified from rat placental conditioned
media using immunoaffinity chromatography exhibit some lactogenic activity at
concentrations above 100 ng/ml. In comparison, recombinant PLP-C purified from a
bacterial expression system shows no significant lactogenic activity in Nb2 lymphoma
bioassays. Furthermore, neither native PLP-B nor PLP-C appear to be the rat homologues
for PLF as factors which stimulate neovascularization of the placenta.
Figure 7- 1 . Effects of native preparations of PLP-B and PLP-C on Nb2 lymphoma
cell proliferation. Cells (5 x 10^), were treated in triplicate wells with varying
concentrations of ovine PRL (D), PLP-B (*) or PLP-C (A) at 37oC for 44 hr.
Cells were harvested after a 4 hr incubation with -^H-thymidine. Mean values +
SEM for two experiments are presented.
Figure 7-2. Effect of recombinant PLP-C on Nb2
lymphoma cell proliferation. Cells (8 x 10''^) were treated in
triplicate with varying concentrations of ovine PRL (•) and
recombinant PLP-C (♦) at 37° C for 44 hr. 3H-thymidine
incorporation was determined after a 4 hr labelling period.
Mean values ± SEM for three experiments are presented.
Figure 7-3. Quantitation of the angiogenic effect of day 18 placental (basal
zone) conditioned medium on retinal endothelial cell migration. Details are as
described in Materials and Methods. Upper wells were loaded with FBS,
serura-free, or placental conditioned medium. Data are expressed as mean ±
SEM of the number of migrating cells from three separate experiments. * P<
0.001 compared to serum-free media (SFM).
Figure 7-4. Quantitation of the angiogenic effect of gestation day 18 placental
(Basal Zone) conditioned media, native PLP-B and native PLP-C on retinal
endothelial cell migration. Details are as described in Materials and Methods.
Upper wells were loaded with BZ medium and PLP-B or PLP-C proteins.
Data are expressed as mean ± SEM of the number of migrating cells from three
separate experiments. * P< 0.001 compared to serum-free media (SFM).
CONCLUSIONS AND FUTURE DIRECTIONS
There has been growing public concern about the abiUty of environmental
contaminants to contribute to adverse effects upon human health. This study grew out of
the desire to investigate the potential mechanisms by which these agents may play a role in
placental growth and development, and uterine disease pathologies, with particular regard
to endometriosis. The objectives of this study were two-fold. Firstly, we investigated the
cellular and molecular processes which could be altered by B(a)P and TCDD, two AhR
agonists, in a human endometrial cell line, RL95-2. Secondly, this study attempted to
characterize the potential role(s) of two placental proteins designated PLP-B and PLP-C
whose expression has been shown to be altered in low birth weight animal models and by
exposure to pNF. The conclusions which may be derived from this study, as well as the
potential directions of future research efforts are described below.
The first part of this study demonstrated that both TCDD and B(a)P were able to
induce the expression of members of the cytochrome P450 family, an action classically
shown to be a consequence of AhR activation (Whitlock, 1993). B(a)P (a major
contaminant in cigarette smoke), but not TCDD, is able to significantly decrease the
expression of immunoreactive EOF receptor protein in RL95-2 cell cultures. A down-
modulation of the EOF receptor has previously been observed after both B(a)P and TCDD
exposure of human keratinocytes (Hudson et al., 1985). Our results indicate that receptor
down-modulation is not directly correlated with CYPlAl induction and it is possible that
reactive metabolites have a role to play in the process, since B(a)P, in contrast to TCDD is
readily metabolized to a series of reactive intermediates able to bind cellular macromolecules
(Gelbion 1980; Leadon et al., 1988).
Furthermore, B(a)P, but not TCDD, was able to significantly decrease the
proliferation of the endometrial adenocarcinoma cells. Endometriosis is an estrogen
responsive disease, and estrogen actions on the uterus may be mediated through growth
factors like EGF (Lingham et al., 1988; Nelson et al., 1991). Our data correlate with the
etiological association of cigarette smoking with a decreased incidence of endometriosis.
The role of reactive metabolites of B(a)P as a potential mechanism of EGF receptor down-
modulation should be examined by analysis of RL95-2 cell cultures for metabolic products.
Furthermore, the use of an antagonist to the AhR would also aid in determining whether or
not the effects observed on the EGF receptor and/or the formation of B(a)P metabolites are
mediated as a result of receptor activation.
This study next evaluated the potential role of genes and gene products which could
contribute to the elaboration of changes in the invasive phenotype of these endometrial cells
as a mechanism of disease promotion. TCDD, but not B(a)P, was observed to increase the
steady state levels of uPA mRNA. Neither compound, however, was able to significantly
alter the fibrinolytic activity of the conditioned medium of treated cells, as evaluated by
fibrin zymography. Similarly, both compounds were able to increase the expression of
TIMP-1 mRNA, but were without effect on TIMP-2 expression levels.
B(a)P, but not TCDD, was able to significantly decrease the overall ability of
treated cells to invade matrigel membranes. The fact that cellular invasiveness was
decreased by B(a)P correlates with epidemiological data regarding this disorder, however,
the TIMP-l data from our study does not. Our interpretation of the invasion data may need
to take into consideration the experimental design of the assay in which the cells invade
through an inverted membrane system. As a consequence of this, the observed inability of
B(a)P treated cells to attach to the matrigel membrane would account for the observed
B(a)P invasion data, an outcome which brings into consideration the role of adhesion
factors in the invasion process. Thus an investigation of the B(a)P effects on potential
alteration in the expression of members of the integrin and cadherin family may be
warranted, presently an area of research which may be gaining momentum in the field of
endometriosis (Lessey et al., 1994; van der Linden et al., 1994). The fact that B(a)P
resulted in a significant growth arrest of our endometrial cell cultures may also contribute to
the observation of reduced invasive activity by these cells. Finally, the overall invasiveness
of these cells may be the net result of alterations in the expression of other members of the
MMP and TIMP families. An investigation of the potential changes in the expression of
these genes and gene products would contribute to a better understanding of the control of
the invasion process in uterine cells.
This part of the study also demonstrated that TCDD was able to significantly
increase the expression of IL-lp and TNF-a, cytokines which potentially could contribute
to the proinflammatory processes observed in endometriosis (Koyama et al., 1993; Ramey
et al., 1993; Rana et al., 1996). The time course of TCDD induction of these responses
may be important in that IL-lp has been shown to increase the secretion of TNFa from
cytotrophoblast cell cultures, albeit without a change in mRNA levels for TNFa (Knofler et
al., 1997). These genes are regulated at a number of levels, especially at the post-
transcriptional level in the case of TNF-a (Tabibzadeh, 1991). Thus it would be
worthwhile to examine whether the observed changes in mRNA are manifested as
increased levels of protein expression for these cytokines.
The fact that TNFa has been shown to alter the ability of cells from primary
endometrial cultures to attach to basement mesothehal cells (Zhang et al., 1993b), as well
as regulate the dyscohesion of epithehal cells (Tabibzadeh et al., 1995), may indicate a role
for this cytokine in the modulation of adhesion factor expression. Furthermore, the
observation of increased levels of prostaglandins in the peritoneal fluid of endometriotic
patients may be the result of cytokine mediation. Prostaglandins have been implicated in
the increase in endometrial vascular permeability which occurs during the implantation
process (Chen et al., 1995). The potential of TCDD and B(a)P treated RL95-2 cultures to
release prostaglandins as a consequence of enhanced cytokine production can also be
The alterations produced by TCDD and B(a)P treatment of the RL95-2 endometrial
carcinoma cells are summarized in Figure 8-1. As outlined, TCDD tends to stimulate what
could be chai-acterized as proinflammatory responses in these cells, while B(a)P produces
decreased growth and decreased invasive potential. Further work, however, needs to be
done to more completely characterize the overall responses of these cells to these
prototypical environmental agents.
The second part of this study attempted to characterize the biological activities of
two proteins secreted by the rat placenta during pregnancy. PLP-B and PLP-C were
successfully purified from the conditioned medium of placental explant cultures using
antibodies generated against the proteins for immunoaffinity chromatography. The native
proteins exhibited molecular weights, pi and immunoreactivity similar to that of the
previously characterized placental proteins (Deb et al., 1991b; Ogilvie et al., 1990b). Both
PLP-B and PLP-C were also expressed as recombinant proteins in mammalian and
bacterial expression systems, respectively. This is the first successful expression of
recombinant PLP-C since attempts to express it in mammalian host systems have been
Evaluation of the biological activity of the native PLP-B and PLP-C preparations
demonstrated that both protein preparations exhibited low levels of lactogenic activity;
however, this lactogenic activity was not observed for the recombinant PLP-C. The lack of
activity by recombinant PLP-C may be a consequence of glycosylation, but this is presently
difficult to determine due to the inability to express PLP-C in mammaUan host systems.
The placental conditioned medium, but not native PLP-B or PLP-C, exhibited significant
angiogenic activity as evaluated by the ability of these preparations to stimulate the
chemotaxis of human retinal endotheUal cells. Future studies could utilize the purified
recombinant proteins in order to localize their binding sites after ['"^I] labeling.
Crosslinking studies could be performed with [^^^I] labeled probe in an attempt to
chai"acterize the receptors for these proteins. The fact that PLP-B and PLP-C are major
secretory placental proteins present during the second half of pregnancy argues for a
physiological role during this period.
, , Endometriosis
Figure 8-1. Comparison of the epidemiological factors in the incidence of endometriosis
with alterations in gene expression in the endometrial cell line RL95-2.
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Grantley Dexter Charles was born on December 15th 1967, in San Fernando,
Trinidad. Despite being raised Baptist he spent his formative middle and high school under
the tutelage of Irish Catholic monks (Presentation Brotherhood) at a parochial Catholic
Grammar School. Upon graduation he worked as a substitute elementary school teacher
for a year before entering the University of the West Indies in St. Augustine, Trinidad in
1987. There, after a three year travail, he received his B.Sc. degree in the Natural Sciences
with a major in Chemistry. After a one semester stint as a middle and high school teacher,
he came to the United States in January 1991, to pursue his then dream of a Master's
Degree in Forensic Science at the John Jay College of Criminal Justice (CUNY) in New
York. His interest being stirred by drug pharmacology and toxicology, he apphed to and
was accepted into the Department of Pharmacology and Therapeutics at the University of
Florida in Fall 1992. There he gravitated to the laboratory of Dr. Kathleen Shiverick where
for the course of his graduate study he has worked in the area of reproductive and
developmental toxicology. He has accepted a postdoctoral position at The Dow Chemical
Company in Midland, Michigan, in the laboratory of Dr. Edward Carney, Project Leader
for the Developmental and Reproductive Toxicology (DART) Group.
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.
Kathleen T. Shiverick, Chair
Professor of Pharmacology
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.
David N. Silverman
Professor of Pharmacology
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.
Thomas C. Rowe
Associate Professor of Pharmacology
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.
4 i V '■ ''
Assistant Professor of Pharmacology
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope an^-qualily^as a^
dissertation for the degree of Doctor of Philosophy.
William C. Buhi
Professor of Biochemistry
and Molecular Biology
This dissertation was submitted to the Graduate Faculty of the College of Medicine
and to the Graduate School and was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.
' Dean, College of Medicine
Dean, Graduate School